The lower sensitivity of in vitro cytotoxicity assays currently restricts their use as alternative to the fish acute toxicity assays for hazard assessment of chemicals in the aquatic environment. In vitro cytotoxic potencies mostly refer to nominal concentrations. The main objective of the present study was to investigate, whether a reduced availability of chemicals in vitro can account for the lower sensitivity of in vitro toxicity test systems. For this purpose, the bioavailable free fractions of the nominal cytotoxic concentrations (EC50) of chemicals determined with a cytotoxicity test system using Balb/c 3T3 cells and the corresponding free cytotoxic concentrations (ECu50) were calculated. The algorithm applied is based on a previously developed simple equilibrium distribution model for chemicals in cell cultures with serum-supplemented culture media. This model considers the distribution of chemicals between water, lipids and serum albumin. The algorithm requires the relative lipid volume of the test system, the octanol-water partition coefficient (K(ow)) and the in vitro albumin-bound fraction of the chemicals. The latter was determined from EC50-measurements in the presence of different albumin concentrations with the Balb/c 3T3 test system. Organic chemicals covering a wide range of cytotoxic potency (EC50: 0.16-527000 microM) and lipophilicity (logK(ow): -5.0-6.96) were selected, for which fish acute toxicity data (LC50-values) from at least one of the three fish species, medaka, rainbow trout and fathead minnow, respectively, were available. The availability of several chemicals was shown to be extensively reduced either by partitioning into lipids or by serum albumin binding, or due to both mechanisms. Reduction of bioavailability became more important with increasing cytotoxic potency. The sensitivity of the Balb/c 3T3 cytotoxicity assay and the correspondence between in vivo and in vitro toxic potencies were increased when the free cytotoxic concentrations instead of the nominal cytotoxic concentrations were used as measure of cytotoxic potency. The few remaining prominent differences between cytotoxic and acute toxic concentrations can be explained by a more specific mechanism of acute toxic action than basal cytotoxicity. It is concluded that the frequently observed low sensitivity of in vitro cytotoxicity test systems, compared to fish acute toxicity assays, at least in part, can be explained by differences in the availability of chemicals in vitro and in vivo. Moreover, neglecting these differences systematically causes a bias of the correlation between in vivo and in vitro toxic potencies of chemicals. Taking them into account, however, increases the predictivity of the in vitro assays.