The present study was conducted to elucidate the possible molecular mechanisms involved in the antispermatogenic activity of l-CDB-4022, an indenopyridine. In this study, 45 day-old male Sprague-Dawley rats were treated with a single oral dose of l-CDB-4022 (2.5 mg/kg) or vehicle, and blood and testes were collected at various time points. The rate of body weight gain was not affected, but a significant loss of testes weight was induced by l-CDB-4022. Serum hormones were assayed using specific RIA's or ELISA', and testicular protein and RNA were analyzed by Western blotting and RT-PCR, respectively. There was a significant decrease in inhibin B and concomitant increase in FSH in serum from l-CDB-4022-treated rats, but serum levels of activin A, testosterone, and LH were unchanged. Western analysis of testicular lysates from l-CDB-4022-treated rats exhibited phosphorylation of ERK1/2 at 4 h and later time points. Loss of nectin/afadin complex occurred at 48 h, but there was an increase in levels of integrin-1, N-cadherin, -catenin, and -catenin protein at 24 h and later time points. Increase in expression of FasL and Fas receptor was detected 8 and 24 h after l-CDB-4022 treatment. The ratio of the membrane to soluble form of stem cell factor (SCF) mRNA was decreased. Immunohistochemical analysis of testicular sections indicated a dramatic disruption of the Sertoli cell microtubule network in l-CDB-4022 treated rats. Collectively, these results suggest that l-CDB-4022 activates the MAPK pathway, reduces expression of prosurvival factors such as the membrane form of SCF, alters expression of Sertoli-germ cell adherens junction proteins, disrupts Sertoli cell microtubule structure, and induces the proapoptotic factor, Fas, culminating in germ cell loss from the seminiferous epithelium.