Sperm motility-initiating substance (SMIS), a novel motility inducer from newt egg-jelly, is activated by the release from associated jelly substances at the beginning of internal fertilization and affects female-stored sperm. We examined motility initiation kinetics of newt sperm in response to SMIS by monitoring the changes of sperm intracellular calcium ([Ca2+]i). In quiescent non-motile sperm loaded with the Ca2+ indicator Fluo-4, intracellular free Ca2+ was observed around mitochondria using confocal scanning laser microscopy. A slight increase in [Ca2+]i occurred simultaneously and transiently at motility initiation in sperm treated with either heated jelly extract (hJE) containing activated SMIS, or a low osmotic solution, which naturally initiates motility in externally-fertilizing amphibians and can initiate motility in urodele sperm. When the increase of [Ca2+]i at motility-initiation was monitored using spectrofluorometry, large increases in [Ca2+]i occurred immediately in the low osmotic solution and within 1.5 min in the hJE. In the intact jelly extract (no heating), small increases of [Ca2+]i irregularly occurred from around 1 min and for about 4 min, during which motility was differentially initiated among sperm. These results indicate that the SMIS induces differential initiation of sperm motility depending on the activational states of the SMIS and its overall activity. The motility initiation in the jelly extract was delayed in sperm whose intracellular Ca2+ had been chelated with BAPTA-AM. The relative levels of [Ca2+]i were variable with a mean of 414 ± 256 nmol/L among resting sperm, suggesting that the level of [Ca2+]i in the resting sperm modulates the responsiveness to the SMIS.