Drosophila melanogaster S2 cells provide attractive features for efficient expression of heterologous gene products. We report the optimization of transfection conditions for transient expression of green fluorescent protein (GFP) in Drosophila S2 cells by lipofectin and electroporation. GFP expression in lipofectin transfection was optimum at a transfection time of 24 h, a 5:1 ratio of lipofectin to DNA, and a seeding density of 4 x 10(5) cells/cm(2). On the other hand, GFP expression from electroporation transfection was optimum at 1125 V/cm, 250 mu F, DNA content of 20 mu g, and a seeding density of 1.0 x 10(7) cells/cuvette. Use of the optimum lipofectin method for transient expression resulted in an approximately 3-fold more efficient transfection than the electroporation method. We also investigated the stable transformation of Drosophila S2 cells by using the lipofectin method. Stably transformed polyclonal cell populations expressing GFP were isolated after 4 weeks of selection by using hygromycin B. The optimum ratio of plasmid pAc5CPPA-GFP to pCoHygro for the cotransfection of cells was 49 : 1 for GFP expression. Recombinant GFP expressed in stably transformed S2 cells was found primarily in the intracellular fraction with a molecular weight of 28 000. The maximum GFP concentration in the S2 cells was approximately 9 mg/l. We demonstrated the expression of GFP in Drosophila S2 cells and its use as a reporter for the analysis of gene expression.