Differentiation in olive callus cultures was induced by changing the plant growth regulator content, particularly 2,4-dichlorophenoxyacetic acid, in the growth media at 25°C. These cultures have been maintained for an extended period with low polyploid nuclei levels. Analysis of olive callus cultures indicated that the acyl lipid composition varied according to the state of differentiation and to incubation temperature. Heterotrophic olive callus was characterized by its ability to accumulate triacylglycerol rich in oleate, a situation comparable to developing olive fruit. In fact, oleate-rich triacylglycerol was enhanced in heterotrophic callus cultured at 35°C. Greening calli, however, were noticeably found to possess typical chloroplastic lipids, indicating the presence of intracellular chloroplastic structures, which was confirmed by electron microscopy. These cultures also exhibited acyl compositions with increased amounts of linoleate and α-linolenate, particularly in chloroplastic lipids, and a corresponding decrease in oleate and stearate. In this study we have shown that olive callus in various stages of differentiation can be cultured by manipulating the plant growth regulator concentration in the growth media. Such morphological changes were further reflected by alterations in acyllipid composition. These cultures have remained viable for extended periods and, therefore, appear to be suitable for further investigations of the regulation of lipid formation.