The mammalian protein SPAG16L, the ortholog of Chlamydomonas Pf20, is an axoneme central apparatus protein necessary for flagellar motility. The SPAG16L protein sequence contains multiple potential phosphorylation sites, and the protein was confirmed to be phosphorylated in vivo. A yeast two-hybrid screen identified the testis-specific kinase, TSSK2, to be a potential SPAG16L binding partner. SPAG16L and TSSK2 interactions were confirmed by coimmunoprecipitation of both proteins from testis extracts and cell lysates expressing these proteins, and their colocalization was also noted by confocal microscopy in Chinese hamster ovary cells, where they were coexpressed. TSSK2 associates with SPAG16L via its C-terminal domain bearing WD repeats. The N-terminal domain containing a coiled coil motif does not associate with TSSK2. SPAG16L can be phosphorylated by TSSK2 in vitro. Finally, TSSK2 is absent or markedly reduced from the testes in most of the SPAG16L-null mice. These data support the conclusion that SPAG16L is a TSSK2 substrate.