Starch gel electrophoresis and vertical flat bed electrophoresis in polyacrylamide gels were used to separate butyrylesterases of pulmonary alveolar macrophages (PAMs) from RF/Al(+) and RF/Al(–) mice. PAM esterases from (+) mice showed five bands in starch and five or six in acrylamide whereas four and three or four bands, respectively, were found in extracts from (–) animals. The additional band or bands present only in positive samples corresponded to the Es-2 prealbumin serum esterase found only in the RF/Al (+) animals. This isozyme was more sensitive to diisopropylfluorophosphate than any other PAM esterase. The serum counterpart was sensitive to the same degree. None of the macrophage esterases were inhibited by eserine sulfate. Spectrophotometric analysis of serum esterase activity indicated a statistically significant increase in female mice from both sublines as compared to their respective males, but did not show a difference between the two sublines. The usefulness of the esterase marker in studies of PAM origin and pathologic conditions is discussed.