A previous report from this laboratory revealed that the newly recognized nonmuscle myosin heavy chain II-C (NMHC II-C) contains an alternative exon composed of 24 nucleotides encoding 8 amino acids. This exon is spliced into loop I of NMHC II-C and is present in a wide variety of tissues and cell lines. Insertion of this exon leads to an increase in the actin-activated MgATPase activity and in vitro motility of heavy meromyosin II-C compared to the noninserted isoform. RT-PCR analysis reveals that the mRNA encoding the inserted isoform is highly expressed in many human epithelial tumor cell lines such as liver, HepG2; prostate, PC-3; pancreas, PANC-1; breast, MCF-7, and lung, A-549 cells. Interestingly, when we quantitated the expression level of all three nonmuscle myosin isoforms (NMHC II-A, II-B and II-C) in tumor cell lines vs normal cell lines, the expression of inserted II-C was elevated (greater than 8-fold) in the tumor cell lines compared to the normal cell lines. In contrast, NMHC II-A and II-B were decreased (greater than 3- and greater than 4-fold, respectively) in the tumor cell lines as determined by scanning immunoblots. Analysis of breast, kidney and lung tumors from humans confirmed the increase in NMHC II-C expression in all three tumors compared to their normal associated tissue. Using siRNA to reduce the inserted isoform by 90% in the A549 lung epithelial cancer cell line leads to 80% growth inhibition at 156 h. This inhibition of growth was almost completely rescued by exogenous expression of the inserted NMHC II-C, but only partially rescued by noninserted II-C. These studies suggest a putative role for the NMHC II-C isoform in certain epithelial tumor cell lines.