Nonmuscle myosins are widely distributed and play important roles in the maintenance of cell morphology and cytokinesis. In this study, we compare the detailed kinetic and functional characterization of naturally occurring transcript variants of the motor domain of human nonmuscle myosin heavy chain (NMHC)-2C. NMHC-2C is alternatively spliced both in loop-1 and loop-2. Isoform 2C0 contains no inserts in either of the loops and represents the shortest isoform. An 8-amino acid extension in the loop-1 region is present in isoforms 2C1 and 2C1C2. Isoform 2C1C2 additionally displays a 33-amino acid extension in the loop-2 region. Transient kinetic experiments indicate increased rate constants for F-actin binding by isoform 2C1C2 in the absence and presence of nucleotide, which can be attributed to the loop-2 extension. ADP binding shows only minor differences for the three transcript variants. In contrast, larger differences are observed for the rates of ADP release both in the absence and presence of F-actin. The largest differences are observed between isoforms 2C0 and 2C1C2. In the absence and presence of F-actin, isoform 2C1C2 displays a 5-7-fold increase in ADP affinity. Moreover, our results indicate that the ADP release kinetics of all three isoforms are modulated by changes in the concentration of free Mg(2+) ions. The greatest responsiveness of the NMHC-2C isoforms is observed in the physiological range from 0.2 to 1.5 mM free Mg(2+) ions, affecting their duty ratio, velocity, and tension-bearing properties.