Peripheral blood lymphocytes (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders. We have systematically compared four retroviral vectors, designed according to different strategies, for their efficiency in transfer and expression in human PBLs of the same reporter gene. The receptor gene used in the study codes for the human low-affinity nerve growth factor receptor (LNGFR), and is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression by immunofluorescence, with single cell resolution. Peripheral blood mononuclear cells (PBMCs), as well as human hematopoietic cell lines of myeloid and lymphoid origin, were transduced with the four vectors and analyzed for efficiency of gene transfer, integration and stability of vector proviruses, and LNGFR expression at both RNA and protein level. Fluorescence-activated cell sorter analysis of coexpression of LNGFR and lineage-specific cell surface markers was performed in transduced cell lines, PBLs, and T- cell clones to study gene expression on specific cell subpopulations. Although crucial differences were observed among different constructs, all retroviral vectors could transduce, under appropriate infection conditions, T-cell populations representative of the normal immune repertoire. Gene transfer and expression could be demonstrated also in circulating progenitors of mature T cells. Expression of the transduced gene was heterogeneous among cell populations infected with the different vectors, with optimal results obtained by two of the four constructs. Finally, we have devised a simple protocol based on vector- mediated gene transfer and positive immunoselection of the transduced cells that produces virtually 100% gene-modified cells. This may represent a crucial improvement in the way of designing efficacious protocols involving the use of gene-modified T lymphocytes in clinical studies.