Differential sensitivity of frog twitch and slow/tonic fibers to Ca²⁺ and Sr²⁺ suggests that these two types of fibers express different troponin C (TnC) isoforms. To date only one TnC isoform from anurans (resembling the mammalian fast-twitch isoform) has been isolated and characterized. In this study we examined the possibility that anuran striated muscle contains more than one TnC isoform. To this end, we determined the TnC isoform composition of 198 single fibers from the rectus abdominis of the cane toad (a mixed slow/tonic and twitch muscle) and of toad cardiac muscle, using a method which enables identification of TnC isoforms based on the effect of Ca²⁺ on their electrophoretic mobility. The fibers were typed according to their myosin heavy chain (MHC) isoform composition. The data indicate that striated muscle of the cane toad contains two TnC isoforms; one (TnC-t) present in all fibers displaying only twitch MHC isoforms, and the other (TnC-T/c) present in fibers displaying the tonic MHC isoform and in cardiac muscle. For a subpopulation of 15 fibers, the TnC isoform composition was also compared with Ca²⁺- and Sr²⁺- activation characteristics. Fibers containing the TnC-T/c isoform were ~3 fold more sensitive to Ca²⁺, ~40 fold more sensitive to Sr²⁺, and responded to a ~4.6 fold broader range of [Ca²⁺] than fibers containing the TnC-t isoform. The Ca²⁺-activation properties of toad fibers containing the TnC-T/c isoform appear to be consistent with the previously reported physiological characteristics of amphibian slow/tonic muscle fibers.