The insulin-like growth factor-binding proteins (IGFBPs) in rat serum were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western ligand or immunoblotting. A ligand blot of adult female rat serum revealed at least 20 proteins that bound labeled IGF-I specifically. They ranged in size from about 24-50K, and their pI values ranged from approximately 5.5-8.0. Immuno- and ligand blots showed that IGFBP-3 appeared as a broad band composed of numerous individual spots of about 40-45K, with pI values ranging from about 5.5-7.7. Immunoblots showed that IGFBP-4 resolved into at least three spots of approximately 29K, with pI values of about 6.3-6.7 and an approximately 24K nonglycosylated form that usually appeared as a single spot with a pI of about 7, but occasionally it resolved into a doublet. Immunoblots of neonatal rat serum using antiserum to IGFBP-2 resulted in immunoreactivity of two sets of proteins: approximately 33K forms composed of two proteins and approximately 30K forms comprising at least five different variants with pI values of about 7.0-7.5. However, ligand analysis showed that only the 30K forms had binding activity. IGFBP-1, -5, and -6 were not detectable by immunoblotting. However, a set of about 29K IGFBPs with pI values of approximately 5.9, which appears in significant amounts in the serum of pups and diabetic rats and in placental tissue-conditioned medium, has been tentatively identified as IGFBP-1 by ligand blotting. One finding of particular interest is an approximately 50K BP with a pI of about 6 that comigrates with IGFBP-3. However, unlike IGFBP-3, which is glycosylated and undergoes proteolysis during gestation, this approximately 50K IGFBP is not susceptible to endoglycosidase-F treatment and persists throughout pregnancy. Immunoblotting analysis revealed weak cross-reactivity between the approximately 50K IGFBP and antiserum to IGFBP-4. These results show that the rat serum IGFBPs are more heterogeneous than was previously realized. The two-dimensional system will allow changes in these different forms to be evaluated critically in different physiological and experimental states.