1. The role of cellular Ca2+ in the transport of glucose has been investigated by determining the time-course of tension development and the release of 45Ca and 3-0-[14C]methylglucose from preloaded rat soleus muscles. 2. Electrical stimulation, 2,4-dinitrophenol (0.05 mM) and hyperosmolarity (200 mM mannitol) were all found to induce a rapid rise in tension and the rate coefficient of 45Ca release, which coincided with an acceleration of 3-0-[14C]methylglucose efflux. 3. Caffeine (10 mM) or exposure to K+ -substituted buffer induced a rapid increase in tension and the release of 45Ca, but a much later stimulation of 3-0-methylglucose efflux. This delayed response may be related to the fact that both factors induce a pronounced suppression of the effect of various agents known to stimulate sugar transport.4. Following a washout period of 120 min at 0 degreesC, the return to 30 degrees C elicited a prompt transient rise in the rate coefficient for the release of 45Ca and 3-0-[14C]meth ylglucose to levels, respectively, 2.8 and 14.6 times the control levels measured at 30 degrees C. The magnitude of these peaks appeared to be a function of the duration of the exposure to 0 degrees C. Cooling also led to a stimulation of the uptake of 3-0-[14C]methylglucose, and phlorizin suppressed the rise. 5. It was not possible to detect any significant effect of insulin on basal tension or on the influx or efflux of 45Ca. However, in a hyperosmolar environment, insulin (10-100 munits/ml) induced a marked further rise in tension, indicating that the hormone can elicit a redistribution of cellular Ca2+. 6. It is concluded that a rise in the cytoplasmic concentration of free Ca2+ constitutes a part of the mechanism by which the glucose transport system is activated by a variety of stimuli, perhaps also insulin.