We studied the production of rhamnolipids by Pseudomonas aeruginosa UFPEDA 614 in submerged culture, using glycerol as the carbon source. A rhamnolipid yield of 15.9 g/L was obtained with 40 g/L glycerol and 5 g/L sodium nitrate as nitrogen source after 7 days of cultivation. Structural analysis carried out at different cultivation periods showed that the four major mono-rhamnolipid homologues are present in higher proportion in the first 48 h. Over time, the corresponding four major di-rhamnolipid homologues predominated, representing about 75 % of the total rhamnolipids after 96 h. Physicochemical analysis of the rhamnolipid mixtures obtained at different cultivation periods showed that the sample obtained from the first day of cultivation had the lower critical micelle concentration (15.6 mg/L), which is probably related to the higher proportion of mono-rhamnolipids. The results presented here show that the composition of the mixture of rhamnolipid homologues produced by P. aeruginosa UFPEDA 614 varies over time and that this variation influences the physicochemical properties of the mixture. These findings can be used in order to produce rhamnolipid mixtures that have suitable properties for different applications.