Spectrins represent a family of membrane-associated proteins responsible for membrane flexibility and cell shape in erythrocytes, and probably in most nonerythroid cells. Spectrin functions as a tetramer consisting of two heterodimers each containing two subunits termed α and β. In humans, αI and αII spectrins but not β spectrins are characterized by the presence of an Src homology 3 (SH3) domain. As a tool to investigate the function of spectrin SH3 domains we derived several monoclonal antibodies (mAb) to the recombinant human αI or αII spectrin SH3 domain. Immunostaining using these monoclonal antibodies indicated expression of αI spectrin in cell bodies and αII spectrin in neurites of granule neurons in mouse primary cerebellar cultures. Monoclonal antibodies reactive to αI spectrin SH3 domain indicated expression of a protein(s) containing an αI-like SH3 domain in cytoplasmic vesicular-like structures in GFAP-positive cells in these cultures. In NIH 3T3 fibroblasts, these antibodies label macropinocytic vesicles. Together, these data and Western blotting results suggest expression of at least three spectrin-SH3 domain antibody-reactive proteins.