We investigated the chronic effects of luteinizing hormone (LH) treatment on adult rat Leydig cell structure and function. Two groups of sexually mature male Sprague-Dawley rats were used; controls and rats implanted subdermally with LH-filled Alzet miniosmotic pumps (delivers 24 micrograms of LH per day). After 2 weeks of LH treatment, testes of these rats were fixed by 2.5% glutaraldehyde in cacodylate buffer and processed and embedded in epon-araldite for light and electron microscopy and electron microscopic immunocytochemistry. Using light microscopic stereology, Leydig cell volume density, number of Leydig cells per testis, and the average volume of a Leydig cell were determined. Additionally, the organelle volumes per Leydig cells were quantified by electron microscopic stereology. Sterol carrier protein-2 (SCP2) and catalase in Leydig cells were immunolocalized via the Protein A gold method. Isolated and purified Leydig cells were used to determine the LH-stimulated (100 ng/ml) testosterone secretory capacity per Leydig cell in vitro and to compare the SCP2 and catalase content in equal numbers of Leydig cells using immunoblot analysis. After 2 weeks of LH-treatment, Leydig cell number per testis and the average volume showed a two-fold increase. All organelles tested, except the lipid droplets, were significantly (P < 0.05) increased two-fold in volume per Leydig cell. Testosterone secretory capacity per Leydig cell was increased approximately six-fold in the LH-treated group. Immunolabeling studies showed that the intraperoxisomal SCP2 content was significantly greater (P < 0.05) and the catalase content was significantly lower (P < 0.05) in LH-treated rats compared to to controls. Immunoblots showed that the total SCP2 content per cell is greater and the catalase content per cell is similar in Leydig cells of LH-treated rats compared to controls. In summary, chronic LH treatment produced hyperplasia, hypertrophy and increased testosterone secretory capacity in leydig cells of adult rats. However, the increase in the testosterone secretory capacity per Leydig cell exceeds the degree of Leydig cell hypertrophy, which cannot be explained by a generalized increase in volumes of all Leydig cell organelles in the LH-treated rats. These results also suggested that chronic LH treatment induces differential synthesis of peroxisomal proteins, i.e. an increase in SCP2 synthesis and no change in catalase synthesis. This resulted in peroxisomes rich in SCP2 and lower in catalase. Significance of these effects in relation to the increased steroidogenic capacity of Leydig cells remains to be determined.