The motor function of vertebrate unconventional myosins is not well understood. In this study, we initiated the baculovirus expression system to characterize a novel myosin I from bovine adrenal gland that we had previously cloned [Zhu, T., & Ikebe, M. (1994) FEBS Lett. 339, 31-36], which is classified as myosin I beta. The expressed myosin I beta was well extracted when calmodulin was coexpressed in Sf9 cells. The recombinant myosin I beta cosedimented with actin in an ATP dependent manner. The purified myosin I beta was composed of one heavy chain and three calmodulins. The electron microscopic image of myosin I beta confirmed its single-headed structure with a short tail, which is similar to that of brush border myosin I (BBMI). Myosin I beta showed high K+,EDTA--ATPase activity (approximately 0.14 mumol/min/mg) and Ca(2+)-ATPase activity (approximately 0.32 mumol/min/mg), and the KCl/pH dependence of these activities was different from that of conventional myosin. Mg(2+)-ATPase activity of myosin I beta alone was increased above pCa 6, while the actin dependent activity was not affected by Ca2+. Actin sliding velocity of myosin I beta in the absence of Ca2+ was 0.3-0.5 microns/s at 25 degrees C, which is much greater than that of BBMI (< 0.05 microns/s). The actin sliding activity was abolished above pCa 6, and the sliding activity was restored when exogenous calmodulin was added in the absence of Ca2+. Within similar Ca2+ concentrations, one of the three calmodulins was dissociated from myosin I beta. The results suggest that Ca2+ dependent association of calmodulin may function as a regulatory mechanism of myosin I beta motor activity and that the motor activity of mammalian myosin I is largely different among distinct myosin I isoforms.