Aerobic heterotrophic microorganisms (AH) play a significant role in carbon cycling in cyanobacterial mats; however, little is known about their abundance, diversity and interaction with cyanobacteria. Using catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), bacterial counts in the mat's oxic layer reached a mean of 2.23 +/- 0.4 x 10(10) cells g(-1). Cultivation of AH yielded strains belonging to Actinobacteria, Bacteroidetes, Firmicutes, Gammaproteobacteria and Haloarchaea. 16S rRNA bacterial sequences retrieved from the mat's oxic layer were related to Bacteroidetes, Chloroflexi and Proteobacteria, whereas archaeal sequences belonged to Crenarchaeota and Haloarchaea. Monocultures of cyanobacteria from the same mat were associated with different AH, although Bacteroidetes were found in most cultures. CARD-FISH showed that Bacteroidetes- and Chloroflexi-related bacteria were closely associated with filaments of Microcoleus chthonoplastes. The growth of an axenic culture of M. chthonoplastes PCC7420 was stimulated on the addition of a filtrate obtained from a non-axenic Microcoleus culture and containing only AH and released substances. In contrast, a similar filtrate from a non-axenic Cyanothece-related culture killed Cyanothece PCC 7418. We conclude that a diverse community of AH exist in close association with cyanobacteria in microbial mats and the interactions between AH and cyanobacteria are species-specific and involve the release of substances.