The subcellular location of diacylglycerol acyltransferase (EC 2.3.1.20) in spinach leaves (Spinacia oleracea L. cv. Longstanding Bloomsdale) was determined after sucrose-gradient centrifugation of tissue homogenates. Enzyme activity was associated primarily with gradient fractions containing oil bodies and intact chloroplasts. Gradient fractions enriched with the endoplasmic reticulum contained insignificant levels of diacylglycerol acyltransferase activity. On the basis of chlorophyll, diacylglycerol acyltransferase activity in crude homogenates was not significantly different from that in intact chloroplasts after Percoll density-gradient centrifugation. Among membrane fractions isolated from hypotonically treated chloroplasts, envelopes contained the greatest diacylglycerol acyltransferase and galactosyltransferase activity. These data demonstrated that chloroplasts were a subcellular site for triacylglycerol biosynthesis in spinach leaves.