We have identified sequential changes in the gene expression pattern of human myeloid leukaemia HL-60 cells during their neutrophil differentiation induced by dimethyl sulfoxide (DMSO) that account for the acquisition of mature neutrophil phenotypic hallmarks. Following 1-day DMSO treatment, HL-60 cells were committed to neutrophil differentiation with loss of their proliferative capacity, and gene expression changes were mostly related to transcription and cell cycle, including up-regulation of inhibitor of DNA binding (Id) genes and down-regulation of cyclins, CDC kinases and MCM proteins. After 3-4-day DMSO treatment, zinc finger proteins 266 and 51 (BCL-6) were dramatically up-regulated, and additional gene expression changes, involving functional and signalling proteins as well as genes involved in RNA processing, apoptosis, and protein degradation, correlated with acquisition of the mature neutrophil phenotype. Our data define changes in the gene expression pattern of a rather restricted number of genes during the differentiation process that seem to regulate neutrophil maturation, providing a molecular basis for the acquisition of neutrophil phenotype. Peripheral blood mature neutrophils showed a qualitatively similar expression profile for these selected genes. These results show changes in gene expression during the transformation of a proliferating leukaemic cell into an end apoptotic-prone cell, which might be of importance to get a molecular insight for the use of differentiation therapy in leukaemia.