The esterases of developing rat lung have been investigated by means of disc gel electrophoresis of lung preparations. The enzymes were analyzed by a method that uses thiol esters as substrates in order to correlate these findings with earlier biochemical and cytochemical studies. The esters, p-nitrophenylthiol acetate and p-nitrophenylthiol butyrate, were hydrolyzed by the enzymes in the presence of copper ions. The resultant copper mercaptide was stained with dithiooxamide and analyzed visually and with a densitometer. Lung preparations from prenatal (days 18 through term), postnatal (through the 4th week) and adult animals were examined for the pattern of esterases and for their sensitivity to such inhibitors as O,O-diethyl O- p-nitrophenyl phosphate (E-600) and diisopropyl phosphorofluoridate (DFP) and to temperature inactivation. The zones of esterase activity increased in number and amount during development except for two bands which decreased markedly during late prenatal development. The timing of these changes correlates with various developmental events in the rat lung.