['Phenyl-chloroethyl ureas (CEUs) are a class of anticancer drugs that mainly react with proteins. Two molecules of this family, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU) induced G(1)/S and G(2)/M cell cycle blocks, respectively. We hypothesised that these observations were linked to a differential protein alkylation pattern.', 'Proteins from B16 cells incubated with [(14)C-urea]-CCEU and [(125)I]-ICEU were compared by 2D-analyses followed by MALDI-TOF identification of modified proteins and characterisation of the CCEU binding. Protein expression was investigated by Western blot analyses and cell cycle data were obtained by flow cytometry.', 'Several proteins (PDIA1, PDIA3, PDIA6, TRX, VDAC2) were alkylated by both ICEU and CCEU but beta-tubulin and prohibitin (PHB) were specifically alkylated by either ICEU or CCEU respectively. Specific alkylation of these two proteins might explain the observed difference in B16 cell cycle arrest in G(2) and G(1) phases respectively. Mass spectrometry studies on the alkylated prohibitin localised the modified peptide and identified Asp-40 as the target for CCEU. This alkylation induced an increased cellular content of PHB that should contribute to the accumulation of cells in G(1) phase.'] Is alkylation of prohibitin by cyclohexylphenyl-chloroethyl urea on an aspartyl residue associated with cell cycle G ( 1 ) arrest in B16 cells?