The role and regulation of insulin-like growth factor binding protein-3 (IGFBP-3) in the ovary is not fully understood. We cloned and determined the sequence of 12257 bp of the pig IGFBP-3 gene that includes 4296 bp of the flanking promoter sequence. The porcine IGFBP-3 promoter sequence shares two highly conserved regions with the human and bovine IGFBP-3 promoters and a mouse DNA clone. The first is a 38 bp region between -1095 and -1058, while the second is a 73 bp region between -63 and +10 of the pig sequence. Projected translation of the open reading frame of our sequence gave a peptide sequence identical to that determined by peptide sequencing but adds 27 amino acids upstream of this sequence and is highly similar to the human, bovine, rat and mouse IGFBP-3 peptides. Using RT-PCR we demonstrated that FSH regulates IGFBP-3 mRNA expression in a biphasic manner, with an early induction (maximal at 3 h) and an inhibition at 24 h post FSH treatment. The inhibition at 24 h was not due to changes in IGFBP-3 mRNA stability. A similar pattern of FSH modulation of the IGFBP-3 gene transcription was demonstrated by the reporter activity of granulosa cells transiently transfected with IGFBP-3 promoter constructs. The site for FSH stimulation of the IGFBP-3 gene was localized to the sequence between -61 and -48 relative to the transcription start site. Regulation of IGFBP-3 transcription by FSH suggests a role for IGFBP-3 in follicular development that may be independent of IGF-1.