Propagation of E1 region replacement adenovirus vectors in 293 cells results in the rare appearance of replication-competent adenovirus (RCA). The RCA genome contains E1 DNA acquired from the 293 cellular genome. The Luria-Delbrück fluctuation test was adapted to measure RCA formation rates. To test if structure affected rate, we measured rates during the production of adenovirus vectors with genomes containing three different expression cassette arrangements. The vectors had different extents of sequence identity with integrated Ad5 DNA of 293 cells and had different distributions of identity flanking the expression cassettes. Empty cassette vector RCA rates ranged from 2.5 × 10−8 to 5.6 × 10−10. The extent of sequence identity was not an accurate RCA rate predictor. The vector with the highest RCA rate also had the least overall sequence identity. To define factors controlling RCA generation, adenovirus vectors expressing E2F family proteins, known to modulate recombination gene expression, and overexpressing the human Rad51 recombination protein were analyzed. Compared to their corresponding empty vectors, RCA rates were not increased but were slightly decreased. Initial results suggested expression cassette orientation and/or transcription direction as potential RCA rate modifiers. Testing adenovirus vectors with identical transgene cassettes oriented in opposite directions suggested that transcription direction was not the basis of these rate differences. Thus, the overall structure and location of the transgene cassette had the largest effect on RCA rate. The RCA fluctuation test should be useful for investigators who require accurate measurements of targeted recombination and the probability of RCA formation during stock production.