The Ca2+ binding proteins in the lumen of intracellular Ca2+ stores differ between muscle and non-muscle cells, indicating a specific role of these proteins in intracellular Ca2+ regulation. Since smooth muscle cells possess both muscle and non-muscle characteristics, we have studied the presence and the differential expression of the muscle-type Ca2+ binding proteins--calsequestrin, sarcalumenin, and the histidine-rich Ca2+ binding protein (HCP)--in several smooth muscle tissues from the pig. Western blot analysis showed that among the smooth muscles studied, the cardiac isoform of calsequestrin is expressed at the highest levels in the stomach. Calsequestrin was present at lower levels in ileum and trachea, whereas this protein was undetectable in aorta and main pulmonary artery. The total amount of calsequestrin in the stomach was estimated to be 20-30-times lower than in the pig heart. Whereas calsequestrin from pig presented the same apparent M(r) in sodium dodecyl sulphate polyacrylamide gels as the well characterized protein from rabbit, the apparent M(r) of both sarcalumenin and HCP was lower in pig than in rabbit. The presence of HCP was demonstrated in pig stomach and ileum, while sarcalumenin was detected only in the stomach. These results demonstrate further biochemical differences between smooth muscle cells of large blood vessels and those of the digestive tract. The present findings on the differential distribution of muscle-type Ca2+ binding proteins are discussed in relation to biochemical and functional differences between these smooth muscle cells.