In this report we have characterized a novel, ubiquitously expressed kinase, Clik1, that is predominantly nuclear and undergoes autophosphorylation. Yeast two-hybrid analysis indicated a highly specific association between Clik1 and CLP-36, which was identified in 36 out of 37 Clik1-interacting clones. CLP-36 is a PDZ-LIM protein that localizes to actin stress fibers in nonmuscle cells and associates with alpha-actinin via its PDZ-domain. The association of CLP-36 with Clik1, in turn, is mediated by the C-terminal part of CLP-36 containing the LIM domain, and association was not noted with the closely related ALP PDZ-LIM protein. Interestingly, the association with CLP-36 led to relocalization of the otherwise nuclear Clik1 kinase to actin stress fibers, where it disrupted the periodic staining pattern of CLP-36. Taken together these results establish the CLP-36 PDZ-LIM protein as an adapter, recruiting the Clik1 kinase to actin stress fibers in nonmuscle cells, and suggest that Clik1 represents a novel regulator of actin stress fibers.