The effect of the testicular toxin mono-(2-ethylhexyl) phthalate (MEHP) on the metabolism of energy-yielding substrates in Sertoli cell-enriched cultures has been studied. MEHP stimulated glucose utilization and oxidation. Stimulation of (14)CO(2) production was greater with [1-(14)C]- than with [6-(14)C]glucose. Oxidation of [1-(14)C]pyruvate and [U-(14)C]acetate to (14)CO(2) was reduced by MEHP treatment in the presence but unaffected in the absence of glucose. MEHP increased the incorporation of radioactivity from [1-(14)C]- and [6-(14)C]glucose but not from [U-(14)C]acetate into fatty acids. MEHP markedly increased the production of lactate by Sertoli cells cultured in the presence of 5.5 mm-glucose and 0.5 mm-pyruvate. Decreasing the glucose concentration reduced this stimulatory effect. In glucose-free medium containing 0.5 or 2.5 mm-pyruvate MEHP had no effect on Sertoli cell lactate production. Thus, MEHP appears to stimulate the utilization of glucose by the Sertoli cell but does not appear to have a direct effect on the conversion of pyruvate to lactate, on the conversion of pyruvate to acetyl CoA or a direct effect on the metabolism of acetyl CoA through the Krebs cycle. Increased glucose utilization appears to be a consequence of the increased metabolism of glucose that occurs through the glycolytic and pentose phosphate pathways. These results suggest that in contrast to its effects on the liver, MEHP does not exert a primary effect on the Sertoli cell mitochondrion.