ALP, CLP-36 and RIL form the ALP subfamily of PDZ-LIM proteins. ALP has been implicated in sarcomere function in muscle cells in association with α-actinin. The closely related CLP-36 is predominantly expressed in nonmuscle cells, where it localizes to actin stress fibers also in association with α-actinin. Here we have studied the expression and functions of RIL originally identified as a gene downregulated in H-ras-transformed cells. RIL was mostly expressed in nonmuscle epithelial cells with a pattern distinct from that of CLP-36. RIL protein was found to localize to actin stress fibers in nonmuscle cells similarly to CLP-36. However, RIL expression led to partially abnormal actin filaments showing thick irregular stress fibers not seen with CLP-36. Furthermore, live cell imaging demonstrated altered stress fiber dynamics with rapid formation of new fibers and frequent collapse of thick irregular fibers in EGFP-RIL-expressing cells. These effects may be mediated through the association of RIL with α-actinin, as RIL was found to associate with α-actinin via its PDZ domain, and RIL enhanced the ability of α-actinin to cosediment with actin filaments. These results implicate the RIL PDZ-LIM protein as a regulator of actin stress fiber turnover.