1. In order to investigate the mechanism whereby changes in external pH (pHo) alter tone in rat mesenteric resistance vessels, we have made simultaneous measurements of tension and intracellular Ca2+ [Ca2+]i. Strips of mesenteric artery were loaded with the Ca(2+)-sensitive indicator indo-1 and superfused with physiological salt solution at pH 7.4 and 37 degrees C. 2. An increase of pHo from 7.4 to 7.9 produced an increase in tension. This was accompanied by an increase in [Ca2+]i in resting and high-K(+)-depolarized vessels. Acidification to 6.9 reduced tension and was associated with a fall in [Ca2+]i. Over the pHi range examined, 6.6-7.9, parallel changes in [Ca2+]i and tension were found in K(+)-activated vessels. 3. In contrast to the relatively slow change in [Ca2+]i, pHi and tension with change of pHo, depolarization produced rapid changes in [Ca2+]i and tension, consistent with a more direct action on Ca2+ mobilization. 4. Reducing the external [Ca2+] below 1 mM produced a pronounced fall in [Ca2+]i and force. Changes in [Ca2+]i, produced by alteration of external [Ca2+] (Cao2+) were used to examine the relation between [Ca2+]i and tension. A linear relation was found. Alteration of pHo to 6.9 or 7.9 did not significantly change this relation. When the tension data were normalized to their own maxima, no shift in the tension-Ca2+ relation occurred, suggesting little or no effect of pH on the Ca2+ sensitivity of force production by the contractile proteins. 5. To determine further whether the changes in [Ca2+]i produced by alteration of pHo could account for all the changes observed in tension, [Ca2+]i was restored to control levels while maintaining an altered pHo. When this was done, restoration of [Ca2+]i led to restoration of force. Thus, in this preparation, the changes in [Ca2+]i produced by altering pHo in depolarized vessels can account for the changes in vascular tone.