Polycystin-2 (PC2) is the product of the second cloned gene (PKD2) responsible for autosomal dominant polycystic kidney disease and has recently been shown to be a calcium-permeable cation channel. PC2 has been shown to connect indirectly with the actin microfilament. Here, we report a direct association between PC2 and the actin microfilament. Using a yeast two-hybrid screen, we identified a specific interaction between the PC2 cytoplasmic C-terminal domain and tropomyosin-1 (TM-1), a component of the actin microfilament complex. Tropomyosins constitute a protein family of more than 20 isoforms arising mainly from alternative splicing and are present in muscle as well as non-muscle cells. We identified a new TM-1 splicing isoform in kidney and heart (TM-1a) that differs from TM-1 in the C terminus and interacted with PC2. In vitro biochemical methods, including GST pull-down, blot overlay and microtiter binding assays, confirmed the interaction between PC2 and the two TM-1 isoforms. Further experiments targeted the interacting domains to G821-R878 of PC2 and A152-E196, a common segment of TM-1 and TM-1a. Indirect double immunofluorescence experiments showed partial co-localization of PC2 and TM-1 in transfected mouse fibroblast NIH 3T3 cells. Co-immunoprecipitation (co-IP) studies using 3T3 cells and Xenopus oocytes co-expressing PC2 and TM-1 (or TM-1a) revealed in vivo association between the protein pairs. Furthermore, the in vivo interaction between the endogenous PC2 and TM-1 was demonstrated also by reciprocal co-IP using native human embryonic kidney cells and human adult kidney. Considering previous reports that TM-1 acts as a suppressor of neoplastic growth of transformed cells, it is possible that TM-1 contributes to cyst formation/growth when the anchorage of PC2 to the actin microfilament via TM-1 is altered.