Basic fibroblast growth factor is a polypeptide mitogen with potential biological roles in angiogenesis, differentiation, and the survival of neurons. To study the expression and subcellular distribution of basic fibroblast growth factor in human retinal pigment epithelium in vitro, affinity-purified antipeptide antibodies were generated against a 15 amino acid sequence in the amino-terminus of this growth factor. Analysis of the cross reactivity and specificity of the affinity-purified antibodies demonstrated no ability to recognize acidic fibroblast growth factor and the ability to label specifically the major known forms of basic fibroblast growth factor in whole-cell lysates of retinal pigment epithelium in vitro. Examination of paraformaldehyde- or glutaraldehyde-fixed pigment epithelium at the light and electron microscopic levels revealed prominent localization of basic fibroblast growth factor to the nucleus and nucleolus. In cells fixed with organic reagents, prominent cytoplasmic staining was noted in addition to the nuclear staining seen in aldehyde fixed cells. Investigation of subcellular fractions by Western blot analysis indicated cytosolic as well as nuclear localization of the basic fibroblast growth factor. These analyses, however, demonstrated that the higher molecular weight forms of basic fibroblast growth factor predominate in the nucleus.