Recent evidence suggests that non-muscle myosin IIB (NMIIB) contributes to smooth muscle contraction. This study was designed to determine the effects of NMIIB on the cross-bridge cycling rate. The cross-bridge cycling rate was investigated using sinusoidal analysis. Frequency analysis revealed two asymptotes in the Bode plot of the data; and the intersection of the asymptotes (corner frequency) was higher for the B(+/-) strain (8.73 +/- 1.10 Hz vs 16.56 +/- 1.26 Hz, P < 0.05), consistent with a higher overall cross-bridge cycling rate in heterozygous NMIIB KO (B(+/-)) vs WT mice. These results demonstrate that because of their long duty cycle, NMIIB cross-bridges act as an internal load on smooth muscle myosin to decrease the overall cross-bridge cycling rate and muscle V(max) during force maintenance. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.