Using polymerase chain reactions and genome walking strategies, adeno-associated virus (AAV)-like capsid genes were isolated from rat and mouse liver genomic DNA, where they are present at <5 copies per cell. These genes define two new species of AAVs since their amino acid sequences are <60% identical to each other or to any other AAV capsid. They are most similar to the AAV-5 and goat AAV capsids. A recombinant vector with the mouse AAV capsid and a lacZ transgene (rAAV-mo.1 lacZ) was able to transduce rodent cell lines in vitro. However, it was not able to transduce eight human cell lines or primary human fibroblasts in vitro. It did not bind heparin and its ability to transduce cells in vitro was not inhibited by heparin, mucin, or sialic acid suggesting it uses a novel entry receptor. rAAV-mo.1 lacZ was 29 times more resistant to in vitro neutralization by pooled, purified human IgG than AAV-2. In vivo, rAAV-mo.1 lacZ efficiently transduced murine ocular cells after a subretinal injection. Intramuscular injection of a rAAV-mo.1 human factor IX (hFIX) vector into mice resulted in no detectable hFIX in plasma, but intravenous injection resulted in high plasma levels of hFIX, equivalent to that obtained from a rAAV-8 hFIX vector. Biodistribution analysis showed that rAAV-mo.1 primarily transduced liver after an intravenous injection. These AAV capsids may be useful for gene transfer in rodents.