Activation of Na(+)-nutrient cotransport leads to increased tight junction permeability in intestinal absorptive (villus) enterocytes. This regulation requires myosin II regulatory light chain (MLC) phosphorylation mediated by MLC kinase (MLCK). We examined the spatiotemporal segregation of MLCK isoform function and expression along the crypt-villus axis and found that long MLCK, which is expressed as two alternatively spliced isoforms, accounts for 97 +/- 4% of MLC kinase activity in interphase intestinal epithelial cells. Expression of the MLCK1 isoform is limited to well differentiated enterocytes, both in vitro and in vivo, and this expression correlates closely with development of Na(+)-nutrient cotransport-dependent tight junction regulation. Consistent with this role, MLCK1 is localized to the perijunctional actomyosin ring. Furthermore, specific knockdown of MLCK1 using siRNA reduced tight junction permeability in monolayers with active Na(+)-glucose cotransport, confirming a functional role for MLCK1. These results demonstrate unique physiologically relevant patterns of expression and subcellular localization for long MLCK isoforms and show that MLCK1 is the isoform responsible for tight junction regulation in absorptive enterocytes.