Gene transfer can be achieved in the adult rat heart in vivo by direct injection of plasmid DNA. In this report we define the spatial and temporal limits of reporter gene expression after a single intracardiac injection. pRSVCAT (100 μg), in which the Rous sarcoma virus long terminal repeat is fused to the chloramphenicol acetyltransferase reporter gene, and pαMHCluc (100 μg), in which the α-cardiac myosin heavy chain promoter is fused to the firefly luciferase gene, were injected into hearts, and reporter gene activities were assayed at various times. Both chloramphenicol acetyltransferase and luciferase were detectable in 100% of the rats from 1 to 7 days, in 60% of the rats from 17 to 23 days, and in 30% of the rats from 38 to 60 days after injection. Reporter gene activity was largely limited to a 1–2-mm region of the ventricle surrounding the injection site. Closed circular DNA was far more effective than linear DNA in transfecting cells in vivo. The relative strengths of three different promoters, Rous sarcoma virus long terminal repeat, α-myosin heavy chain, and α1-antitrypsin, all fused to the luciferase reporter gene were determined. The constitutive viral promoter was ∼20-fold more active than the cardiac-specific cellular promoter, and the liver-specific cellular promoter was not active at all in the cardiac environment. Thus, direct injection of genes into the heart offers a simple and powerful tool with which to assess the behavior of genes in vivo. However, the potential of the technique to effect a phenotypic change in the heart is currently limited by the temporal and geographic extent of transfection.