Their functions and localisation can expose gill cells to volume changes. To maintain their vital functions, these gill cells must regulate their own volume after cellular swelling or shrinkage. Recently, we showed that rainbow trout pavement gill cells in primary culture have the capacity to regulate their own volume after cellular swelling induced by hypotonic shock. This so-called regulatory volume decrease (RVD) is associated with intracellular calcium increase, which occurs as a transient peak followed by a plateau when maintained a hypotonic condition. Return to an isotonic medium restores baseline [Ca2+](i) level. In this study, the effect of different xenobiotics on cellular swelling induced RVD and its calcium signal was investigated in trout pavement gill cells in primary culture. These cells were exposed to different pollutants after confluent epithelium was obtained. After 36 h in xenobiotics exposure in vitro, cellular volume and intracellular calcium concentration were measured. Nonylphenol poly- and di-ethoxylate were lethal at concentrations of 10 and 100 muM, respectively. With 10 muM of the diethoxylate form, cells did not die but, unlike non-treated cells, burst during hypotonic shock (2/3rd strength Ringer solution). With 1 muM nonylphenol polyethoxylate (NPnEO), RVD and [Ca2+](i) were reduced. Copper (10 and 100 muM) had no significant effect on gill cell volume regulation. However, the heavy metal modified calcium response to hypotonic shock by inhibiting return to baseline level under isotonic conditions. 10 muM prochloraz and 2,4-dichloroaniline had no effect on cell morphology, volume and [Ca2+](i) concentration. With 100 muM, however, prochloraz was lethal and dichloroaniline increased baseline [Ca2+](i). These results indicate that the effects observed on gill cells are consistent with the known toxic properties of the molecules tested, thus confirming the validity of primary culture to investigate the toxic effects of xenobiotics on fish gill epithelium.