cDNA clones encoding a novel insulin-like growth factor-binding protein (IGFBP) purified from rat serum and human bone cell-conditioned medium have been isolated from rat liver and human placenta, liver, and ovary cDNA libraries. The deduced amino acid sequences of the cDNAs revealed a mature polypeptide consisting of 233 amino acids for the rat, while the human structure contains an additional four-amino acid sequence in the middle region of the molecule. This protein, now proposed to be named IGFBP-4, contains two extra cysteines compared with the previously characterized IGFBP-1, -2, and -3, but the alignment of the remaining 18 cysteines is conserved across the four IGFBPs. Amino acid sequence comparison among the four binding proteins within the rat species demonstrated that both the amino- and carboxy-terminal one thirds of the molecules are highly conserved, while the middle one third region, where no cysteines are present except for the two that exist in IGFBP-4, is the most divergent. The overall sequence homology among the four rat IGFBPs is very similar (53-59%), suggesting that their individual genes diverged from a single ancestral gene at about the same evolutionary time point. Northern analysis of the IGFBP-4 mRNA in rat tissue demonstrated that transcription of the IGFBP-4 gene is highly active in the liver, although a single 2.6-kilobase IGFBP-4 mRNA band was detectable in all tissues examined, including adrenal, testis, spleen, heart, lung, kidney, liver, stomach, hypothalamus, and brain cortex.