We have identified for the first time the presence of chloride intracellular channel (CLIC) proteins in bovine epididymal spermatozoa. CLIC1 was discovered during microsequencing of proteins that co-purified with protein phosphatase 1, PP1gamma2, in sperm extracts. In addition to CLIC1, Western blot showed that two additional CLIC family members, CLIC4 and CLIC5, are also present in spermatozoa. CLIC fusion proteins, GST-CLIC1, GST-CLIC4 and GST-CLIC5, were all able to bind to PP1gamma2 in sperm extracts during pull-down assays. Immunofluorescence microscopy revealed that each of the three isoforms occupies a distinct location within the cell. Given that PP1gamma2 is a key enzyme regulating sperm motility, PP1gamma2-binding proteins, such as the CLIC proteins, are likely to play significant roles in sperm function.