It has been shown previously that acidic K18 and K19 keratins display a differential immunohistochemical pattern of expression during sexual differentiation of the gonads in the rat (Fridmacher et al. (1992) Development 115, 503-517). The present results indicate that K18 and K19 gene expression is regulated at the transcriptional level. The analysis was performed by Northern Blot, reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization. PCR products were cloned, sequenced and used as species-specific K18 and K19 riboprobes for in situ hybridization. K19 mRNA but not K18 mRNA was detected in undifferentiated gonads and in somatic cells of ovarian cords throughout the fetal ovary development. K18 mRNA expression appeared in male gonads, at 13.5 days of gestation, at the onset of testicular differentiation, as the first Sertoli cells differentiated and aggregated to form seminiferous cords. As testicular differentiation progressed, K19 mRNA disappeared and, from 14.5 days of gestation on, fetal Sertoli cells expressed exclusively K18 mRNA. The changes in the transcriptional activity of K19 and K18 genes, observed in male gonads, occur characteristically at the very beginning of testicular differentiation. In the male pathway of sexual differentiation, the switch in K19/K18 gene expression is, in addition to the activation of the anti-Müllerian hormone gene, the most precocious regulative event occurring after the expression of the testis determining factor SRY.