A variety of methods have been successfully employed for the transfection of eukaryotic cells in culture; methods principally used for adherent cell populations such as fibroblasts, epithelial cells and muscle cells. Haematopoietic cells, however, have proven to be quite recalcitrant to transfection by calcium phosphate precipitation, DEAE-dextran and protoplast fusion. The method described-adapted from two previous reports for the transfection of chicken embryo fibroblasts and Chinese hamster ovary cells (1, 2)-uses polybrene/DMSO for the effective transfection of adherent and non-adherent myeloid cells. 1. Adherent mveloid cells: a) Cells grown until three-quarters confluent in DMEM plus 10% fetal bovine serum (FBS). b) Medium replaced with growth medium plus polybrene (30j±g) and DNA (1-lOgg). c) Cells incubated (6 hr, 37 C, 5% CO2) with twice gentle rocking. d) DNA/polybrene solution replaced by 30% DMSO in growth medium (3 min, room temperature). e) Medium aspirated, cells washed twice with growth medium and incubated in growth medium alone. 2. Non-adherent myeloid cells: a) Cells grown to 0.5-1 x 106/ml in RPMI plus 10% FBS. b) Polybrene/DNA added as above. c) Cells harvested by centrifugation (1,000 rpm, 5 min). d) Cells resuspended at 2-3x original concentration in 30% DMSO in growth medium in test tube (1 min, room temperature). e) 5x volume growth medium added to dilute DMSO, cells washed twice by centrifugation and then resuspended at 0.5-1 x 106/ml in growth medium. In both cases, after 48 hr, growth medium was replaced by selective medium and outgrowth of resistant cells observed after 2-4 weeks. These protocols were derived using transfection of a Moloney Murine Leukemia Virus Long Terminal Repeat-bacterial neomycin reistance gene construct. This gene was successfully introduced and stably expressed in the adherent murine monocytic J774 cell line and the non-adherent human promyelocytic HL60 cell line. Other (nonselectable) genes have also been introduced and expressed by this procedure. Typical transfection efficiencies were 10-5 to 10-6 transfectants/jg DNA (-2 logs lower than fibroblast lines). By comparison, calcium phosphate precipitation yielded no detectable transfectants using the myeloid cell lines. The variable which we have found most crucial in this procedure is the time of DMSO shockthis should be determined for each cell type by analysing cell viability at 1, 2 and 3 min or, substituting 15% glycerol if DMSO is found to be toxic.