ABSTRACT. A proteome analysis of the promastigote stage of the trypanosomatid parasite Leishmania infantum (MON-1 zymodeme) is described here for the first time. Total protein extracts were prepared at early logarithmic and stationary phases of replicate axenic cultures and processed by 2D electrophoresis (pH 3–10). A total of 28 differentially regulated proteins were identified by matrix-assisted laser desorption/ionization-tandem time of flight mass spectrometry. This approach has revealed that the electron transfer flavoprotein (ETF) and the eukaryotic elongation factor 1α (eEF1α) subunit have the same differential expression pattern at the protein and mRNA levels, up-regulation in the stationary phase. A low-molecular-weight isoform and an alternatively processed form of the eEF1α subunit have been detected. A 51 kDa subunit of replication factor A is up-regulated in dividing logarithmic promastigotes. None of the proteins described here shows opposite differential regulation values with the corresponding mRNA levels. Taken together with previous approaches to the proteome and the transcriptome, this report contributes to the elucidation of the differential regulation patterns of the ETF, the eEF1α subunit, the 40S ribosomal protein S12, α-tubulin and the T-complex protein 1 subunit γ throughout the life cycle of the parasites from the genus Leishmania.