Previous studies have proposed that increasing the temperature around the rat isolated ileal muscles induces a heat-released substance (HRS) which causes a decrease in the contractile response. Therefore, we have studied various characteristics of this proposed HRS in animals exposed to heat-stress conditions. In these animals, the contraction of muscles in response to carbachol was decreased by increasing the temperature from 37 degrees C to 40 degrees C. Further, bathing the ileum in a conditioned medium prepared by incubation of the ileal muscle at 40 degrees C has shown less contraction at 40 degrees C than in presence of normal Krebs medium. In addition, conditioned mediums, prepared at 40 degrees C were run on SDS-polyacrylamide gel electrophoresis and have shown the presence of a distinct protein band at a m.w. region of 55 kDa. These results confirm our previous studies that increasing the temperature around the muscles induces the HRS that causes the decrease in the contractile response.