Peripheral benzodiazepine (BDZ) receptor (PBR) and diazepam-binding inhibitor/acyl-CoA-binding protein (DBI/ACBP) characterized as a ligand at central BDZ receptors, at PBR with involvement in the regulation of steroidogenesis, and as an intracellular acyl-CoA transporter, are both known to interact with BDZ in adult systems. We investigated their expression after prenatal exposure to BDZ. Diazepam (1.25 mg/kg per day s.c.) was administered to time-pregnant Long Evans rats from gestational day (GD) 14 to 20. Expression of mRNAs encoding for PBR and for DBI/ACBP was studied in the same animals with (33)P-labeled 60 mer oligonucleotides (oligos) by in situ hybridization at GD20, and with (32)P-labeled oligos by Northern blot in steroidogenic and immune organs at postnatal day (PN) 14 and in adult offspring. Prenatal diazepam increased DBI/ACBP mRNA expression in male fetal adrenal and in fetal and PN14 testis. Thymus exhibited increased DBI/ACBP mRNA in male fetuses and in adult female offspring, and reduced organ weight at PN14 in both sexes. In female spleen, an increase in DBI/ACBP mRNA and a decrease in PBR mRNA was seen at PN14. Apart from the finding in spleen, no drug-induced changes in PBR mRNA were observed. The effects of prenatal diazepam were superimposed on treatment-independent sex differences in DBI/ACBP mRNA and PBR mRNA expression. Our data indicate that expression of DBI/ACBP mRNA in steroidogenic and immune organs can be affected by exposure to BDZ during ontogeny, while PBR mRNA expression appears to be less sensitive. They further reveal marked sex differences in the developmental patterns of the two proteins during pre- and postpubertal ontogeny.