c-Cbl-associated protein (CAP) is an SH3-containing adapter protein that binds to the proto-oncogene c-Cbl. Recent work suggests that signaling through these molecules is involved in the regulation of insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Skeletal muscle is the major site of insulin-stimulated glucose disposal but there have been no reports of CAP function in this tissue. Using RT-PCR of mouse skeletal muscle RNA, we discovered a novel splice variant of CAP (CAPSM; GenBank Accession No. AF521593) that is different from the adipocyte form by inclusion of a novel 168 bp fragment. This fragment encodes a peptide sequence that shows very high similarity with exon 25 of the human homologue of CAP (SORBS1). To understand the function of CAPSM in glucose uptake regulation, L6 myotubes were transfected with either CAPSM or a truncated CAPSM devoid of all three SH3-binding domains (CAPDeltaSH3), which prevents CAP association with c-Cbl. Transfection with CAPDeltaSH3 decreased insulin-stimulated 2-deoxyglucose (2-DG) uptake and reduced c-Cbl phosphorylation. In contrast, transfection of L6 myotubes with CAPDeltaSH3 had no effect on dinitrophenol (DNP)- or hypoxia-stimulated glucose uptake, stimuli that work through insulin-independent mechanisms for the regulation of glucose uptake. These data demonstrate the existence of a novel CAP isoform expressed in skeletal muscle, and suggest the involvement of the CAP/Cbl pathway in the regulation of insulin-stimulated glucose uptake in L6 myotubes.