Excitation-contraction coupling in smooth muscle involves activation of myosin light chain (MLC) phosphorylation, which increases activity of the myosin actin-activated ATPase, resulting in contraction. Phosphorylation of MLC phosphatase (SMPP-1M) by Rho-associated kinase or endogenous SMPP-1M-associated kinase inhibits SMPP-1M, enhancing MLC phosphorylation and contraction. However, the precise identity of SMPP-1M-associated kinase remains unclear. Biochemical evidence strongly supports the idea that SMPP-1M-associated kinase is related to the human serine/threonine leucine zipper-interacting protein kinase (hZIPK), which is important in cell apoptosis, and the SMPP-1M-associated kinase has therefore been called ZIP-like kinase (MacDonald, J. A., Borman, M. A., Murani, A., Somlyo, A. V., Hartshorne, D. J., and Haystead, T. A. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 2419-2424). Whether the vascular smooth muscle SMPP-1M-associated kinase is a truncated version of hZIPK, native hZIPK, or a unique homologue of hZIPK is unclear. Here we show that only native hZIPK mRNA and protein are detectable in human vascular smooth muscle cells (VSMCs). High stringency screening of a human aortic cDNA library for the SMPP-1M-associated kinase identified 18 positive clones, all of which proved to be clones of hZIPK. PCR-based studies of VSMC RNA revealed native hZIPK transcripts but no evidence for splice variants of hZIPK or a ZIP-like kinase. Northern blotting studies of multiple vascular and non-vascular tissue RNAs, including human bladder RNA, showed only 2.3 kb of mRNA predicted for full-length hZIPK. Immunoblotting showed native full-length 52-kDa hZIPK expression in VSMCs. Full-length and N-terminal hZIPK bound the C-terminal domain (amino acids 681-847) of the myosin binding subunit (MBS) of SMPP-1M. hZIPK immunoprecipitated with the MBS of SMPP-1M and dominant negative RhoA inhibited the hZIPK-MBS interaction. These data identify hZIPK as the unique SMPP-1-associated kinase expressed in human vesicular smooth muscle and support a role for Rho in promoting the hZIPK-MBS interaction.