We have sought a transfection strategy which preserves cell viability and achieves sufficient efficiency to perform reporter gene assays in primary cultures of thioglycollate-elicited murine peritoneal macrophages. Murine peritoneal macrophages were transfected with an eukaryotic expression vector containing the Rous sarcoma virus enhancer-promoter upstream of the bacterial chloramphenicol acetyl-transferase reporter gene (FC4-CAT). Transfection using DEAE-dextran followed by 10% DMSO provided much higher CAT activity than either calcium-phosphate or lipofection. The transfected macrophages increased CAT activity (1.9-7.6-fold) following stimulation with 10% serum. DEAE-dextran/DMSO-mediated transfection provides a simple, inexpensive method to transfect primary cultures of adherent macrophages with heterologous plasmid DNA. Transfection of macrophages with a CAT reporter gene using this method permits the characterization of gene regulation in primary macrophage cultures in vitro.