A total of 171 untrained, unacclimatized, and unanesthetized rats were either exercised to exhaustion at one of four ambient temperatures (5, 20, 26, or 30 degrees C), or were restrained and heated at an ambient temperature of 41.5 degrees C until their core temperatures reached a preselected end point between 41.0 and 43.3 degrees C. The serum levels of creatine phosphokinase (CRK) and two transaminases (SGOT and SGPT) were determined at 30 min, 24, 48, 72, and 96 h posttreatment. Peak enzyme activity for CPK was noted primarily at the 30-min sampling period and at 24 h for the transaminases. The data indicated that under these conditions a) the transaminase SGOT was elevated in the serum as a consequence of the extent and duration of prior hyperthermia, b) the transaminase SGOT was released in moderate amounts after exhaustive exercise but reached its greatest activity levels following hyperthermia, and c) the activity of CPK was increased by the duration of exhaustive exercise and was less sensitive than either transaminase to prior hyperthermia. As a result, each of the three experimental conditions: a) exercise without hyperthermia, b) exercise with hyperthermia, and c) sedentary hyperthermia, produced a unique pattern of serum enzyme activity that would appear useful in diagnosing a variety of heat- and/or work-induced disorders.