Fowl sperm motility was measured by altering the extracellular pH (pHe) at 30°C and 40°C. At 30°C, the motility of intact spermatozoa was vigorous in a medium in the wide pHe range of 7.3–10.1. In contrast, intact spermatozoa were almost immotile at 40°C in medium below a pHe of 8.1. However, the motility could be restored by increasing the pHe; maximum motility was obtained in medium at pHe 9.4. Stimulation of the motility of demembranated spermatozoa at 40°C was also observed with an increased pHe. However, demembranated spermatozoa at 40°C that had been stimulated by increasing the pHe lost their motility when 1 mmol CaCl2 l−1 was added. Motility was restored by the subsequent addition of 2 mmol EGTA l−1. At a high pHe at 40°C, the flagellar ATPase activity of crude dynein extract was not affected, regardless of the addition of CaCl2 or EGTA. The intracellular pH (pHi) of intact spermatozoa, estimated by measuring the accumulation of 9-aminoacridine fluorescence, increased with increasing pHe at both 30°C and 40°C. These results demonstrate that the reversible temperature-dependent immobilization of fowl spermatozoa at 40°C is inhibited by an increased pHi. Furthermore, it is possible that the effects of the increased pHi may not act directly on dynein ATPase activity, but are mediated by a Ca2+-related substance(s) on the axoneme.