Adeno-associated virus (AAV) vectors are a practical choice for gene transfer, and demand for them is increasing. To cope with the necessity in the near future, we have developed a number of approaches to establish packaging cell lines for the production of AAV vectors. In our previous study, a highly regulated expression of large Rep proteins was obtained by using the Cre-loxP switching system. Therefore, in the present study, to regulate Cap expression as well, we developed an inducible expression system for both Rep and Cap proteins by using an additional set of mutant loxP sequences. The mutants possess two base alterations in the spacer region of loxP and recombine specifically with the same counterpart in the presence of Cre. By using two separate plasmids, one with mutant and the other with wild-type loxP sequences, the expression of two different proteins can be induced simultaneously by Cre recombinase. When the LacZ-encoding plasmid vector was used as a packaging model, a significant packaging titer of 2.1 x 1010 genome copies per 10-cm dish was obtained. These results indicate the importance of controlling Cap expression, in addition to Rep, to achieve an optimum production rate for AAV vectors.