Microsomes isolated from developing seeds ofHelianthus annuuswere prepared in a medium which ensured that endoplasmic reticulum (ER)-bound polysomes remained attached to the ER during homogenization. The microsomes were then incubated with the substrates necessary to sustain the synthesis of triacylglycerols (TAGs). Microsomes that contained high activities of the enzymes involved in the synthesis of TAGs (the enzymes of the Kennedy pathway) accumulated TAGs synthesizedin vitro, resulting in a decrease in their buoyant density. These light membrane fractions could therefore be separated on discontinuous sucrose density gradients from microsomes containing low activities of the enzymes of the Kennedy pathway. Analysis of the microsome fractions by 1H-NMR spectroscopy showed that the TAGs synthesized in the microsomesin vitrowere tumbling isotropically in an environment similar to that of the TAGs in oil bodies. Western blot analysis revealed that microsomes which synthesized large amounts of TAGsin vitrowere also substantially enriched in oleosins. In addition, labelling studies indicated that the oleosins newly synthesizedin vitroby ‘run-on' translation of ER-bound polysomes also localized to light membrane fractions. This indicates that oleosins are specifically enriched in regions of the ER involved in the biogenesis of the oil body.