Diacylglycerol acyltransferases (DGATs) catalyse the final step of the triacylglycerol (TAG) biosynthesis of the Kennedy pathway. Two major gene families have been shown to encode DGATs, DGAT1 (type-1) and DGAT2 (type-2). Both genes encode membrane-bound proteins, with no sequence homology to each other. In this study, the molecular cloning and characterization of a type-2 DGAT cDNA from olive is presented. Southern blot analysis showed that OeDGAT2 is represented by a single copy in the olive genome. Comparative transcriptional analysis revealed that DGAT1 and DGAT2 are developmentally regulated and share an overall overlapping but distinct transcription pattern in various tissues during vegetative growth. DGAT2 is highly expressed in mature or senescing olive tissues. In flowers, the expression of DGAT1 was almost undetectable, while DGAT2 transcripts accumulated at the later stages of both anther and ovary development. Differential gene regulation was also detected in the seed and mesocarp, two drupe compartments that largely differ in their functional roles and mode of lipid accumulation. DGAT1 appears to contribute for most of the TAG deposition in seeds, whereas, in the mesocarp, both DGAT1 and DGAT2 share an overlapping expression pattern. During the last stages of mesocarp growth, when TAGs are still accumulating, strong up-regulation of DGAT2 but a marked decline of DGAT1 transcript levels were detected. The present results show overlapping gene expression for olive DGATs during mesocarp growth, with a more prominent implication of DGAT2 in floral bud development and fruit ripening.