We have isolated human and rat clones of the LIM motif-containing protein kinase, termed LIMK-2. LIMK-2 is related to the neuronally expressed LIM-kinase, whose hemizygous deletion appears to result in cognitive impairment in patients with Williams syndrome. The hallmark of this protein family is the presence of 1 or 2-terminal LIM motifs and an atypical C-terminal protein kinase domain. LIMK-2 mRNA was detected by Northern blot analysis in human tissues, most abundantly in placenta, lung, liver, and pancreas, and also in a variety of cell lines including neuronal, glioblastoma, and mammary carcinoma lines. The LIMK-2 transcript was also induced upon neuroectodermal differentiation of mouse P19 embryonal carcinoma cells. A 65 kDa recombinant LIMK-2 protein was identified in 293 cells stably transfected with a LIMK-2 expression vector. An in vitro kinase assay demonstrates LIMK-2 is autophosphorylated and exhibits serine/threonine kinase activity towards the exogenous substrate MBP. The endogenous 65 kDa LIMK-2 protein was detected in a variety of cell lines, and coprecipitates with a 140 kDa tyrosine phosphorylated protein, but was not itself tyrosine phosphorylated. At the subcellular level, LIMK-2 is localized in both the nucleus and in a Triton X-100 soluble fraction.