Recombinant adeno-associated virus 2 (rAAV2) has been shown to deliver genes to neurons effectively in the brain, retina, and spinal cord. The characterization of new AAV serotypes has revealed that they have different patterns of transduction in diverse tissues. We have investigated the tropism and transduction frequency in the central nervous system (CNS) of three different rAAV vector serotypes. The vectors contained AAV2 terminal repeats flanking a green fluorescent protein expression cassette under the control of the synthetic CBA promoter, in AAV1, AAV2, or AAV5 capsids, producing the pseudotypes rAAV2/1, rAAV2/2, and rAAV2/5. Rats were injected with rAAV2/1, rAAV2/2, or rAAV2/5 into selected regions of the CNS, including the hippocampus (HPC), substantia nigra (SN), striatum, globus pallidus, and spinal cord. In all regions injected, the three vectors transduced neurons almost exclusively. All three vectors transduced the SN pars compacta with high efficiency, but rAAV2/1 and rAAV2/5 also transduced the pars reticulata. Moreover, rAAV2/1 showed widespread distribution throughout the entire midbrain. In the HPC, rAAV2/1 and rAAV2/5 targeted the pyramidal cell layers in the CA1-CA3 regions, whereas AAV2/2 primarily transduced the hilar region of the dentate gyrus. In general, rAAV2/1 and rAAV2/5 exhibited higher transduction frequencies than rAAV2/2 in all regions injected, although the differences were marginal in some regions. Retrograde transport of rAAV1 and rAAV5 was also observed in particular CNS areas. These results suggest that vectors based on distinct AAV serotypes can be chosen for specific applications in the nervous system. © 2004 The American Society of Gene Therapy.