The occurrence of NADP -dependent malic enzymes (NADP -MEs) in several Rhodococcus strains was analysed. The NADP -ME number in Rhodococcus genomes seemed to be a strain-dependent property. Total NADP -ME activity increased by 1.8- and 2.6-fold in the oleaginous Rhodococcus jostii RHA1 and Rhodococcus opacus PD630 strains during cultivation under nitrogen-limiting conditions. Total NADP -ME activity inhibition by sesamol resulted in a significant decrease of the cellular biomass and lipid production in oleaginous rhodococci. A non-redundant ME coded by the RHA1_RS44255 gene located in a megaplasmid (pRHL3) of R. jostii RHA1 was characterized and its heterologous expression in Escherichia coli resulted in a twofold increase in ME activity in an NADP -dependent manner. The overexpression of RHA1_RS44255 in RHA1 and PD630 strains grown on glucose promoted an increase in total NADP -ME activity and an up to 1.9-foldincrease in total fatty acid production without sacrificing cellular biomass. On the other hand, its expression in Rhodococcus fascians F7 grown on glycerol resulted in a 1.3-1.4-foldincrease in total fatty acid content. The results of this study confirmed the contribution of NADP -MEs to TAG accumulation in oleaginous rhodococci and the utility of these enzymes as an alternative approach to increase bacterial oil production from different carbon sources.