The protein product of the novH oncogene, a member of the CCN family, is structurally related to the insulin-like growth factor (IGF) binding proteins (IGFBPs). We have characterized aspects of structure, function, and distribution of this protein, which, as IGFBP-related protein 3 (IGFBP-rP3), is a proposed member of the IGFBP Superfamily. Affinity cross-linking experiments performed with baculovirus synthesized recombinant human IGFBP-rP3 established that rhIGFBP-rP3 binds IGF-I, IGF-II, and insulin with low affinity. Specificity of binding was shown by competitive cross-linking experiments; binding to IGF-I and -II was also demonstrated by nondenaturing Western ligand blots. Northern blot analysis indicated the presence of IGFBP-rP3 messenger RNA (mRNA) in a broad range of human tissues. Western immunoblotting studies, using a polyclonal rabbit anti-rhIGFBP-rP3 antibody, demonstrated that IGFBP-rP3 protein is synthesized in vitro by several breast and prostate cancer cell lines: Hs578T, PC3, P69, and LNCaP cells. Western immunoblotting studies of human biological fluids identified that IGFBP-rP3 was present in normal serum, pregnancy serum, serum from patients with growth hormone receptor deficiency, cerebrospinal fluid, amniotic fluid, peritoneal fluid, and follicular fluid, while IGFBP-rP3 fragments were identified in cerebrospinal fluid, amniotic fluid, and prepubertal and pubertal urine samples. Our studies demonstrate that IGFBP-rP3 exhibits IGF binding, albeit at low affinity, and IGFBP-rP3 thus merits inclusion in the IGFBP Superfamily. The low affinity IGF binding suggests that IGFBP-rP3 may act primarily independently of the IGFs. The synthesis of IGFBP-rP3 by several malignant cell lines and its presence in human biological fluids suggest that this protein possesses other interesting roles, potentially in cell growth regulation.