1. Intracellular pH (pHi) and contraction have been measured simultaneously in isolated longitudinal rat myometrium. Tissue was loaded with the pH-sensitive fluorophore carboxy-SNARF and superfused with oxygenated, buffered salt solution at 37 degrees C and pH 7.4. 2. Small (0.04 pH unit) transient intracellular acidifications followed each spontaneous uterine contraction. The pHi transients lagged contraction by 15 s. Removal of external Ca2+ abolished both spontaneous contractions and the pHi changes. 3. Increasing uterine contraction by depolarization (40 mM K+) or agonist (50 microM carbachol) produced a larger acidification (0.18 pH unit). 4. The acidification with contraction does not appear to be due to increased glycolysis as no increase in lactate efflux occurred with high-K+ stimulation. 5. Uterine contraction is known to be sensitive to changes in pHi. Comparable pHi changes to the pHi transients seen during spontaneous contraction were induced by application and withdrawal of weak acids and bases. The resulting pHi changes (0.02-0.08 pH unit) produced effects on contraction frequency and amplitude, alkalinization augmenting and acidification depressing contractile activity, suggesting that the pHi transients with contraction are functionally important.