Calcium (Ca2+)-mediated signaling is fueled by two sources for Ca2+: Ca2+ can enter through Ca2+ channels located in the plasma membrane and can also be released from intracellular stores. In the present study the intracellular Ca2+ release channel/ryanodine receptor (RyR) from zebrafish skeletal muscle was characterized. Two RyR isoforms could be identified using immunoblotting and single-channel recordings. Biophysical properties as well as the regulation by modulators of RyR, ryanodine, ruthenium red and caffeine, were measured. Comparison with other RyRs showed that the zebrafish RyRs have features observed with all RyRs described to date and thus, can serve as a model system in future genetic and physiological studies. However, some differences in the biophysical properties were observed. The slope conductance for both isoforms was higher than that of the mammalian RyR type 1 (RyR1) measured with divalent ions. Also, inhibition by millimolar Ca2+ concentrations of the RyR isoform that is inhibited by high Ca2+ concentrations (teleost alpha RyR isoform) was attenuated when compared to mammalian RyRs. Due to the widespread expression of RyR these findings have important implications for the interpretation of the role of the RyR in Ca2+ signaling when comparing zebrafish with mammalian physiology, especially when analyzing mutations underlying physiological changes in zebrafish.