1. The effect of lowering the temperature to near freezing-point upon the contractions and [3H]-inositol phosphate responses to carbachol were investigated in longitudinal smooth muscle from the guinea-pig ileum. 2. The peak amplitude of the contraction to a single application of 100 microM carbachol was the same at 37 degrees C and temperatures near freezing-point. However, the sensitivity to carbachol was reduced upon lowering the temperature and the time to peak contraction was increased from 5-10 s to 2-10 min. Even when the temperature was maintained near freezing-point, washing off carbachol produced a relaxation and eventual return of tension to basal levels. 3. Incubating the tissue in 140 mM K+, calcium-free solution or in calcium channel antagonists significantly reduced the carbachol-induced contraction to 10-30% of the control at 37 degrees C and also at 3 degrees C. Thus the majority of the activator calcium required for contraction entered the tissue via voltage-dependent calcium channels (VDCs) at both 37 degrees C and 3 degrees C. 4. The contractions produced by high potassium solutions were less at temperatures close to freezing-point than those at 37 degrees C suggesting that voltage-dependent calcium entry was inhibited as the temperature was lowered. 5. A small part of the contractile response to 100 microM carbachol was resistant to the removal of extracellular calcium at both 37 degrees C and 3 degrees C and this component was increased under depolarizing conditions. This suggests that the release of stored calcium contributes to a minor degree to contraction at both 37 degrees C and 3 degrees C.6. Although 100 microM carbachol produced a statistically significant rise in several [3H]-inositol phosphate isomers at both 37 degrees C and 3 degrees C, the production of [3H]-inositol phosphates was less at 3 degrees C than at 37 degrees C and the increase in their production caused by carbachol was much slower.7. These results suggest that the carbachol-induced contraction at 3 degrees C utilizes both calcium entry through VDCs and calcium release from intracellular stores, as at 37 degrees C. The components of the responses dependent upon intracellular calcium release at 37 degrees C and at temperatures near freezing-point were similar. However, the production of [3H]-inositol phosphates, including the calcium-mobilizing second messenger inositol (1,4,5) trisphosphate (Ins(1,4,5)P3), is reduced at such low temperatures.