Little is known about the occurrence and linkage between secreted insecticidal virulence factors in natural populations of Bacillus thuringiensis (Bt). We carried out a survey of 392 Bt strains isolated from various samples originating from 31 countries. The toxicity profile of the culture super-natants of these strains was determined individually against Anthonomus grandis (Coleoptera) and Spodoptera littoralis (Lepidoptera). We analyzed ␤-exotoxin I production and searched for the genes encoding Vip1-2, Vip3, and Cry1I toxins in 125 of these strains. Our results showed that these insecticidal toxins were widespread in Bt but that their distribution was nonrandom, with significant linkage observed between vip3 and cry1I and between vip1-2 and ␤-exotoxin I. Strains producing significant amounts of ␤-exotoxin I were more frequently isolated from invertebrate samples than from dust, water, soil, or plant samples. Although microbial populations display apparent overall genotypic and phenotypic homogeneity, many microbial species exhibit considerable diversity, which has an impact on the biological properties of individual strains [16]. This is especially important for microorganisms with pathogenic properties, such as Bacillus thuringien-sis (Bt). Bt, which has a worldwide distribution [19], is a sporogenic bacterium used in biological control that synthesizes specific insecticidal toxins [24]. To date, most studies on Bt insecticidal toxins have focused on ␦-en-dotoxins, which are produced as parasporal inclusions, whereas little is known about the pathogenic potential of the compounds secreted into the culture medium [7, 15]. Many Bt strains secrete vegetative insecticidal proteins (Vips) during vegetative growth [27]. Two classes of Vip toxins have been described. The first consists of a binary system composed of two proteins, Vip1 and Vip2, which are 100 kDa and 52 kDa in size, respectively. These proteins are highly toxic to certain coleopteran species [27, 28]. The second class consists of an 82.5-kDa protein , Vip3, which is active against a wide spectrum of lepidopteran insects [8]. These two classes of protein do not display sequence homology. Cry1I (also referred as to CryV in the literature) is another insecticidal toxin secreted during early stationary phase. It is active against certain lepidopteran and coleopteran insect larvae [15]. Finally, several Bt strains also produce a thermostable exotoxin, known as ␤-exotoxin I or thuringiensin [7]. This compound is an adenine nucleotide analogue, highly toxic to a wide range of insect species [9], that is thought to inhibit RNA polymerase [25], thereby affecting insect molting and pupation, in some cases having teratogenic effects [5]. ␤-exotoxin I is also toxic to mammalian cells [3, 18] and is very persistent in the environment [4]. It has therefore been banned for public use in accordance with WHO recommendations [29]. If we are to improve the efficacy and ensure safety of Bt products, we need to increase our understanding of the genetic variability of natural Bt populations with respect to the expression of these secreted virulence factors. In this study, we analyzed the toxicity of culture superna-tants of natural Bt isolates to larvae of Spodoptera litto-Correspondence to: V. Sanchis;