Diacylglycerol kinase activity is found in both adult and fetal lung. Approximately 27 and 52% of the total activity is found in microsomes and cytosol, respectively. The activity is maximal at pH 7.4. The apparent Km for ATP is 0.11 mM and 0.21 mM for cytosol and microsomes, respectively. The apparent Km for dioleoylglycerol is 0.05 mM for cytosol and 0.14 for microsomes. Maximal activity in cytosol and microsomes is obtained with 2.0 mM dexoycholate. Other detergents cannot substitute for deoxycholate. Phosphatidylglycerol stimulates activity in the absence and in the presence of deoxycholate. Phosphatidylserine also stimulates activity, whereas phosphatidylethanolamine was inactive and phosphatidylcholine inhibited the reaction. Linoleic acid produced inhibition. The general properties of the enzyme were similar for fetal and adult lung. Diacylglycerol kinase from microsomes and cytosol fraction from both fetal and adult lung was most active with dioleoylglycerol and diacylglycerol from egg phosphatidylcholine. Significantly lower activity was obtained with dipalmitoylglycerol. Phosphatidylglycerol did not alter the relative substrate preferences. The activity in microsomes increased with development from 19 days gestation to a maximal activity at 21 days gestation. Maximal activity was about 2-fold higher than the adult. The activity dropped rapidly reaching adult values prior to birth (22 days gestation). The activity in cytosol fractions increased gradually from 19 days gestation, reaching adult values by 22 days gestation.