The insulin-responsive R3230AC mammary tumor possesses type I and type II insulin-like growth factor (IGF) receptors, and membrane preparations display affinity cross-linking of 125I-labeled IGF-I to IGF-binding proteins (IGFBPs). To identify the IGFBPs produced, Northern blotting analysis of poly(A)+ RNA extracts from tumor tissue was performed. Although transcripts of IGFBP-1 were not detected, intense bands were obtained at 1.7 and 6 kilobases (kb) when hybridized with radiolabeled IGFBP-2 and IGFBP-5 cDNA probes, respectively. A 2.6-kb band and a 2.4-kb weaker band were observed after hybridization with IGFBP-3 and IGFBP-4 probes, respectively. When IGFBP-6 cDNA was used, two bands were seen: a higher mol wt band at 6.3 kb and a smaller one at 1.3 kb. Tumors from streptozotocin-induced diabetic rats displayed an increase in the expression of IGFBP-2, and insulin treatment for 3 days normalized the IGFBP-2 mRNA levels. Tumors from diabetic rats displayed no change in IGFBP-3, -4, -5, and -6 mRNA levels from tumors of normoglycemic rats. However, tumors from insulin-treated rats showed significantly higher levels of mRNA for IGFBP-4 and IGFBP-5 than tumors from normoglycemic or diabetic rats. A similar, but less pronounced, pattern of changes in IGFBP-3 mRNA was seen, whereas levels of IGFBP-6 mRNA were unchanged throughout. To identify the cell type producing the mRNAs for these IGFBPs, in situ hybridization of tissue sections was used. Procedures were established that localized three of the five IGFBPs expressed in this tumor tissue. This technique showed that IGFBP-3 mRNA transcripts were observed mainly in endothelial cells of tumor vasculature, although they were also detected in stromal cells, IGFBP-4 was present mainly in tumor stroma cells, and IGFBP-5 mRNA was expressed predominantly in the epithelial cells of this tumor. Expression of IGFBP-5 mRNA transcripts was significantly diminished in primary and long term cultured R3230AC cells grown in alpha-Minimum Essential Medium and 10% fetal bovine serum. Tumors arising from injection of long term cultured cells that were injected into isologous rats contained high amounts of mRNA transcripts for IGFBP-5, suggesting the presence, in vivo, of positive regulators for the expression of this BP. Tumor cells cultured in the presence of insulin displayed a 2.2- to 2.5-fold increase in the expression of IGFBP-5. These findings imply a role for insulin as a regulator of the expression of IGFBP-5 in the R3230AC adenocarcinoma.