In vivo assessment of toxicant action on Leydig cell function is subject to homeostatic mechanisms which make it difficult to determine whether any changes seen in serum testosterone (T) concentration are due to extragonadal endocrine alterations or to a direct effect on the Leydig cell. For example, metal cations administered in vivo have been shown to depress serum T concentration and alter serum concentrations of pituitary hormones in laboratory animals. The studies reported here use a testicular cell culture technique to evaluate Leydig cell testosterone biosynthesis in the presence of several metal cations. To determine the site of toxic action, the Leydig cells were stimulated to produce testosterone by using human chorionic gonadotrophin (hCG), dibutyl cyclic adenosine monophosphate (db-cAMP), or several substrates required for the biosynthesis of testosterone. hCG was chosen because resultant T production requires an intact membrane receptor and db-cAMP was used to test for post LH receptor defects caused by the metals. The other substrates were chosen to isolate the effect of metals on enzymatic pathways. Collagenase dispersed testicular cells (15% Leydig cells) were incubated with metal cations (1 to 5000 μm) for 3 hr in the absence and presence of maximally stimulating concentrations of hCG, db-cAMP, 20α-hydroxycholesterol (HCHOL), or pregnenolone (PREG), and T concentration was determined by radioimmunoassay. In one separate experiment we also tested the effect of the substrates progesterone, 17α-hydroxyprogesterone, and androstenedione on Cd²⁺-treated Leydig cells. The results show no change in Leydig cell viability with any metal cation treatment during the 3-hr incubation. Ca²⁺, Cr³⁺, Fe³⁺, Mg²⁺, Na⁺, or Pb²⁺ had no effect on stimulated testosterone. Dose-response depression in both hCG- and db-cAMP-stimulated T production were seen with Cd²⁺, Co²⁺, Cu²⁺, Hg²⁺, Ni²⁺, and Zn²⁺ treatment. Surprisingly, Cd²⁺, Co²⁺, Ni²⁺, and Zn²⁺, which caused a depression in hCG- and db-cAMP-stimulated T production, caused significant increases in HCHOL- and PREG-stimulated T production over untreated and similarly stimulated cultures. This indicates that these cations may act at multiple sites within the Leydig cell.