Gene therapy for acute cardiac events such as myocardial infarction requires early gene expression over an entire region of myocardium, which has not been possible using adeno-associated virus (AAV) vectors to date. Here we demonstrate marked improvement in the distribution and rapidity of gene expression in myocardium using the AAV pseudotype 6 (AAV6) vector, compared to the standard serotype 2 (AAV2) vector. An alkaline phosphatase (AP) reporter construct driven by the chicken beta-actin promoter was packaged in either AAV6 or AAV2 capsids and delivered to rat hearts in vivo by direct injection. AP expression was evident in both AAV6 and AAV2 vector-treated hearts as early as 1 day after injection, but increased rapidly in AAV6 vector-treated hearts during the first 7 days. The amplitude of AP activity produced by the AAV6 vector was 5-fold greater than that produced by the equivalent AAV2 vector at both 3 and 7 days postinjection. Additionally, the AAV6 vector transduced a myocardial volume that was 10-fold larger than the AAV2 vector. These results indicate the significant potential of AAV6 serotype vectors for early gene expression and widespread regional transduction of myocardium, both auspicious results for in vivo applications in acute cardiac disease.