An efficient, simple, and reproducible DNA-mediated gene transfer procedure has been developed for primary cultures of adult rat hepatocytes. Calcium phosphate-DNA precipitate is formed in complete culture medium during 5 hr incubation with cells. Unabsorbed precipitate is then washed out, and 40 hr later gene expression is measured. Under optimal conditions, up to 20-25% of cells in cultures transfected with the beta-galactosidase (lacZ) gene stain positively for this activity, and cells transfected with the chloramphenicol acetyl transferase (CAT) gene, fused to a strong promoter, express CAT activities of 10-14 nmoles/min per mg protein. Five conditions were optimized based on transfection efficiency, CAT expression, and cell viability. (i) Medium composition: the presence of protein, such as fetal bovine serum or bovine serum albumin, in the medium was essential. (ii) Cell substratum: tissue culture plastic was superior to calf skin collagen and Matrigel. (iii) Cell density: 0.5-1.0 X 10(6) cells/60-mm dish were superior to higher densities. (iv) Duration of exposure to calcium phosphate-DNA: 5-8 hr was better than shorter or longer times. (v) Length of time hepatocytes were maintained in culture before initiating transfection: 2-3 days was superior to earlier times. This procedure was successful with reporter genes linked to three different eukaryotic promoters. These included a chimeric promoter containing the polycyclic aromatic hydrocarbon-responsive enhancer of the cytochrome P450c gene (CYP1A1), which was shown to confer upon the CAT gene responsiveness to polycyclic aromatic hydrocarbons comparable to that of the native P450c gene. This transfection procedure should be of considerable use for the study of liver-specific gene expression in primary hepatocyte cultures.