Abstract Extracts containing mature Rhizopus oryzae lipase overexpressed in Pichia pastoris (rROL) and commercial ones from the native microorganism (nROL) have been characterized. The specific activity of rROL extract was more than 40-fold higher than nROL. The presence of multiple bands of lipases around 34 kDa was detected by western blot and zymogram analysis in both extracts. Nevertheless, rROL showed a slightly lower molecular weight than nROL. The presence of hydrolytic activity not recognised as derived from a lipase was also detected in nROL at higher molecular weights. The influence of the ionic strength on lipase activity was assayed and there was an effect both on pH and optimal temperature. Some differences were found between the two extracts. The specificity against triacylglycerol esters and p-nitrophenol substrates was also analyzed. Similar behaviour was noted towards triacylglycerol esters; however rROL showed opposite behaviour compared to nROL towards p-nitrophenol esters with preferences for long chain derivatives. The properties of rROL were partially different from those reported for nROL showing the influence of the pre-pro-sequence of ROL, the post-translational modifications of Pichia pastoris and the effect of an esterase in nROL powder.