A highly sensitive assay for determination of infectious titers of recombinant adeno-associated virus (AAV) by limiting dilution analysis is described. This assay is capable of detecting single infectious events and can therefore provide an absolute rather than relative measure of infectivity. The assay utilizes a HeLa-derived AAV2 Rep/Cap-expressing cell line, D7-4, grown in 96-well plates and infected with replicate 10-fold serial dilutions of AAV2 vectors in the presence of adenovirus type 5. Forty-eight hours after infection, vector genome replication is determined by quantitative PCR (Q-PCR). A linear relationship between vector genome input and replicated copy number (slope = 2670 copies per vector genome) was determined, enabling detection of one infectious event per well by Q-PCR. The observed binomial distribution of the end-point data confirmed that single infectious events could be detected, and allowed calculation of infectious titers by the Kärber method. Analysis of an AAV2 reference vector, AAV-hFIX16, in 21 independent determinations gave an average ratio of AAV vector genomes (VG) to infectious units (IU) of 8.3 +/- 4.2 VG/IU, a value close to the theoretical limit. No significant differences in vector particle-to-infectious unit ratios were observed between vectors purified by column chromatography (9.3 +/- 5.0 VG/IU, n = 7) and cesium chloride gradient ultracentrifugation (6.4 +/- 3.2 VG/IU, n = 7).