The rat stomach fundus is enriched with the 5-hydroxytryptamine (5-HT)2B receptor, a recently cloned subtype of the 5-HT2 receptor family. Unlike other members of the 5-HT2 receptor family, the 5-HT2B receptor in the rat stomach fundus was not coupled to phosphatidylinositol (PI) hydrolysis. The purpose of this study was to characterize further the signal transduction mechanism of the 5-HT2B receptor in rat stomach fundus. Nitrendipine (1 microM) inhibited the maximal contraction to 5-HT (10 microM) by approx. 60%. 5-HT contractions were inhibited by approximately the same magnitude in the absence of extracellular calcium as in the presence of nitrendipine, indicating that calcium influx through voltage-dependent calcium channels accounted fully for the dependence of the 5-HT contraction on extracellular calcium. Depletion of both extracellular calcium and intracellular calcium stores abolished 5-HT contraction. Ryanodine (30 microM), an inhibitor of calcium release from internal stores, inhibited significantly the nitrendipine-insensitive 5-HT contraction, suggesting that this component of the contraction was due to calcium release from a ryanodine-sensitive site. Bisindolylmaleimide (5 microM), a specific inhibitor of protein kinase C (PKC), inhibited 5-HT contraction in either the absence or presence of nitrendipine, suggesting that activation of PKC is also important. Taken together, these data indicate that the 5-HT2B contractile receptor in the rat stomach fundus is coupled to calcium influx through voltage-dependent calcium channels, intracellular calcium release, and activation of PKC. These actions may reflect a novel coupling mechanism unrelated to increases in PI hydrolysis.