The feasibility of gene marking or gene therapy protocols making use of purified CD34+ cells greatly depends on the efficiency of stable transduction. The great potentiality of umbilical cord blood CD34+ cells prompted us to evaluate which factor(s) play a major role(s) in influencing the transduction efficiency of retroviral vectors. CD34+ cells, isolated with an immunomagnetic device (MiniMacs, Miltenyi Biotech, Germany), 90-95% pure as assessed by cytofluorimetric analysis, were transduced with the LXSN retroviral vector carrying the neomicine resistance gene. Infection was made with virus-containing supernatant of Am12 env+ packaging cell line, having a titer of 2×106 PFU/ml. All the infections were performed using a multiplicity of infection of 10 PFU/cell. Multiple or single infections were performed with the following schedules: i) up to 6 infection lasting one hour over 3 days; ii) a 72 hr infections substituting fresh viral supernatant and cytokines every 24 hrs; iii) a single 72 hr infection. In most experiments, CD34+ cells were preincubated with different cytokine cocktails for 24 or 48 hrs before infection. Efficiency of transduction was evaluated both by clonogenic assays and semiquantitative PCR analysis performed either immediately or after 7 day expansion in the presence of cytokines and G-418. G-418-resistant CFU-E, BFU-E and CFU-GM colonies were scored after 7 and 14 days. We provided evidence that the efficiency of transduction was mainly dependent on cell preincubation with growth factors rather than on the number of infections. Precisely, infection performed on freshly isolated CD34+ was almost ineffective (0.4%) while 48 hr preincubation in the presence of SCF*+IL-6+IL-3 allowed to reach a level of transduction up to 40% regardeless of the number of infections. In this respect it is noteworthy that a single infection, lasting as long as a multiple infection course gave identical results. In conclusion the results obtained suggest that high efficiency of transduction of umbilical cord blood CD34+ cells can be achieved in liquid, stroma free, cultures.