Abstract The mechanism through which Cr(VI) reduces the intracellular levels of ATP in hamster fibroblasts (BHK 21 line) were investigated. Leakage of pool components during treatment of cells prelabelled with [ 3H]adenosine was examined. Adenylate pool composition was analyzed by thin-layer chromatography (TLC) in cells incubated with [ 3H]adenosine immediately after treatment with Cr(VI), and the endogenous concentrations of different nucleotides were determined by high-performance liquid chromatography (HPLC). Interference of Cr(VI) with different steps of ATP biosynthesis could be assessed. Treated cells shown increased loss of radioactive hypoxanthine and inosine in the treatment medium, but no nucleotides were detected outside the cells. A marked unbalance of the endogenous adenylate pool was produced by chromium, consisting in a strong decrease of ATP, accompanied by a very pronounced increase of ADP and AMP. Treatment with Cr(VI) caused an altered distribution of label among the different adenylate pool components, especially an accumulation of radioactivity in the ADP and AMP fractions. Also other effects on the soluble pool were detected by HPLC analysis, namely the reduction of NAD content and variations in th guanylate pool which closely paralleled the ones observed in the adenylate pool. Energy charge values, calculates on the basis of HPLC and TLC data, allowed to detect a specific inhibition of ADP phosphorylation to ATP. An interference with the early steps of adenylate synthesis could be inferred from the enchanced amounts of hypoxanthine and ionsine in the treated cultures.