In this study we investigated the effects of 4-chloro-3-ethylphenol on Ca2+ homeostasis, and we report that this compound induces Ca2+ release from a ruthenium red-sensitive Ca2+ release channel present in skeletal muscle terminal cisternae. When tested with bovine cerebellar microsomes, the Ca(2+)-releasing activity of 4-chloro-3-ethylphenol was reduced by ruthenium red and unaffected by heparin. In PC-12 cells, HL-60 cells, human fibroblasts, the human hepatoma cell line PLC/PRF/5, and Jurkat cells, 4-chloro-3-ethylphenol released Ca2+ from intracellular thapsigargin-sensitive stores. Although decreased, its effect was retained after treatment of intact/permeabilized cells with inositol-1,4,5-trisphosphate (IP3)-mobilizing agonists/IP3, whereas pretreatment of permeabilized cells with ruthenium red reduced the Ca(2+)-releasing activity of 4-chloro-3-ethylphenol. These results provide functional evidence for the presence of a Ca2+ channel distinct from the IP3 receptor, having pharmacological similarities to the ryanodine receptor, in the intracellular Ca2+ stores of a variety of nonexcitable cells. We also demonstrate that a monoclonal anti-ryanodine receptor antibody recognizes a protein in human fibroblasts with similar apparent molecular mass the ryanodine receptor. Thus, the intracellular Ca2+ stores of mammalian cells appear to be endowed with two distinct intracellular Ca2+ channels.