S-100a0 protein, the alpha alpha isoform of the S-100 family, stimulates basal (Mg2+-activated) adenylate cyclase (AC) activity associated with the sarcolemma, longitudinal tubules and terminal cisternae of rat skeletal muscle cells. The stimulatory effect of S-100a0 on AC activity is maximal around 5 microM S-100a0 and half-maximal around 0.2 microM S-100a0. Also, the stimulatory effect is greatest on the AC activity associated with the terminal cisternae than on the other membrane fractions studied. These data are discussed in relation to the subcellular localization of S-100a0 in muscle cells.