An in vitro inhibin bioassay method based on suppression of the FSH content in cultured rat anterior pituitary cells was applied to charcoal-treated testicular extracts of normal and bilaterally cryptorchid rats. The validity of the method was established from the statistical evidence of parallelism between dose-response curves of the rat testicular extract (RTE) and an ovine testicular lymph reference preparation. From 10 bioassays, an average index of precision of 0.11 and an interassay coefficient of variation of 16.3% were obtained. The specificity of the bioassay in the measurement of RTE was based on the following criteria: (1) the suppression of pituitary cell FSH content, but not LH; (2) the activity was not due to cytotoxic effects of RTE in the bioassay system,as assessed by a 51Cr release cytotoxicity test; (3) the activity was absent from kidney and liver extracts; (4) the active material in RTE was a proteinaceous macromolecule with a molecular weight above 17,000, as determined by gel filtration; and (5) no evidence of any synergistic or antagonistic factors in RTE. After the induction of cryptorchidism, testicular inhibin levels were significantly (P < 0.001) reduced after 1 week, reaching 10% of the levels in normal animals by 6 weeks. Serum concentrations of FSH and LH in cryptorchid animals were significantly (P < 0.05) elevated by 3 days and reached levels 3–4 times those found in corresponding sham-operated controls by 6 weeks. In relating the testicular inhibin content of these animals with serum concentrations of FSH or LH, a significant (P < 0.001) inverse correlation (r = -0.74 for FSH and r = -0.60 for LH) was obtained. These results are consistent with the hypothesis that inhibin is involved in the physiological feedback regulation of pituitary FSH secretion.