The ability of megakaryoblastic leukemia 1 (MKL1) to function as a serum response factor (SRF) coactivator is regulated through its association with G-actin. In the cytoplasm, MKL1 binds to G-actin through RPXXXEL (RPEL) motifs. However, dissociation of MKL1 from G-actin triggers its translocation into the nucleus where it stimulates SRF-mediated gene expression. Previous characterization of rat MKL1 gene products has identified several isoforms: full-length MKL1, basic, SAP, and coiled-coil domain (BSAC), MKL1-elongated derivative of yield (MELODY), and MKL1met. In this study, we have investigated whether these MKL1 isoforms, which contain different numbers of RPEL motifs, differ in their subcellular localization, transcriptional activity, and effect on dendritic number and axonal length. Immunofluorescent staining of cultured cortical neurons expressing individual FLAG-tagged MKL1 isoforms indicated that all MKL1 isoforms are present in both the cytoplasm and the nucleus. However, MKL1met, which contains two RPEL motifs, shows enhanced nuclear staining compared with the other three isoforms, full-length MKL1, basic, SAP, and coiled-coil domain, and MKL1-elongated derivative of yield, which contain three RPEL motifs. Consistent with its preferential nuclear localization, overexpression of MKL1met, but not other isoforms, increases SRF-mediated transcriptional responses and reduces the number of dendrites. In contrast to the inhibitory effect of MKL1met on dendritic number, axonal length is not affected by overexpression of any of the MKL1 isoforms. These findings suggest that the subcellular localization of MKL1 isoforms, which is mediated by the number of actin-binding RPEL motifs, regulates their effect on SRF-mediated gene expression and dendritic morphology.