The insulin receptor substrate 1 (IRS-1) contains at least 11 sequence motifs that are rich in proline (P), glutamic acid (E), serine (S), and threonine (T), i.e., PEST regions. Proteins with PEST regions turn over rapidly. IRS-1 is degraded rapidly in vivo upon exposure of 3T3-L1 adipocytes to insulin. The intracellular, calcium-dependent, neutral proteases known as calpains are one possible mechanism by which IRS-1 may be degraded. To begin to investigate this possibility, purified exogenous calpain was shown to degrade IRS-1 in cell-free extracts from basal and insulin-treated cells and rat recombinant IRS-1 in vitro. Only two proteolytic fragments could be detected. One had a mol wt of approximately 79 kDa, arising from the C-terminus end, and the second had a mol wt of approximately 90 kDa arising from near the N-terminus, possibly a product of the same cleavage event, since the mol wt of IRS-1 from insulin-treated cells was approximately 170 kDa. These results suggest that IRS-1 may serve as a substrate for calpain in vivo, accounting for its rapid degradation.