Adeno-associated viral (AAV) vectors promote long-term gene transfer into muscle in many animal species. Increased expression levels may be obtained by using alternative serotypes in combination with repeated administrations. Here we compared AAV vectors based on serotypes 1, 2 and 5 in immunocompetent mice and assessed the feasibility of multiple administrations of either identical (readministration) or different (cross-administration) serotype-based vectors. A 1-year-long dose-response study confirmed the superiority of recombinant (r)AAV1, achieving transduction levels 5 to 10-fold higher than rAAV2 and rAAV5 in mouse skeletal muscle, respectively. Repeated administration demonstrated that increased gene transfer level was achieved with a second injection of rAAV1 following the first administration of rAAV2 or rAAV5. A readministration study with a vector encoding a different gene allowed the evaluation of gene expression from the second vector only. All three rAAVs were inhibited when the animals were previously exposed to the same serotype. In contrast, no significant change in gene expression from the second vector was observed in cross-administration. A humoral immune response was elicited against the viral capsid for all three serotypes following the initial exposure. Neutralizing antibody (NAB) levels correlated with the vector dose injected. No significant cross-reactivity of NAB from a given serotype toward another was observed in vitro. These data provide the first direct comparative evaluation of re- and cross-administration of rAAV1, rAAV2 and rAAV5 in muscle, and further indicate that rAAV1 is capable of transducing muscle tissue when cross-administered.