Previous work has demonstrated that the presence of an alternatively-spliced exon in loop 1 near the ATP-binding region of the myosin II heavy chain results in an increase in both the actin-activated MgATPase activity and in vitro motility of vertebrate smooth muscle and nonmuscle heavy meromyosins (HMMs) II-B and II-C. We now report that the presence of a 21 amino acid insert in loop 2, which is near the actin-binding site of nonmuscle myosin heavy chain II-B, results in the loss of these activities. This alternative exon is normally expressed in certain neuronal cells, such as the Purkinje cells in the cerebellum and has been shown to affect motor coordination in mice. Surprisingly, the Vmax of baculovirus-expressed HMM II-B2 (inserted isoform) was less than 7% of that of the noninserted HMM II-B0 (Vmax: 0.01 +/- 0.0003 s-1 for HMM II-B2 compared to 0.15 +/- 0.01 s-1 for the HMM II-B0; n=4 for each) and no significant movement was observed in the in vitro motility assay for HMM II-B2. Cosedimentation assays in the presence and absence of ATP showed that the affinity of HMM II-B for actin is not affected by the insertion of the 21 amino acid residues into loop 2. To study the activity of nonmuscle myosin II-B2 in cells, we investigated the ability of full-length GFP-myosin II-B to rescue multinucleation. In COS-7 cells, decreasing nonmuscle myosin II-B0 using siRNA results in multinucleated cells due to a defect in cytokinesis. Introducing GFP-myosin II-B0 into the II-B depleted cells significantly reduced multinucleation. In contrast, transfection of GFP-myosin II-B2 into the II-B siRNA-treated cells failed to rescue multinucleation. Therefore, insertion of these 21 amino acids into loop 2 of nonmuscle myosin II-B causes a loss of activity both in vitro and in cells.