We have previously described substantial variation in the level of expression of two linked genes which were introduced into transgenic petunia plants using Agrobacterium tumefaciens. These genes were (i) nopaline synthase (nos) and (ii) a chimeric chlorophyll a/b binding protein/octopine synthase (cab/ocs) gene. In this report we analyze the relationship between the level of expression of the introduced genes and T-DNA structure and copy number in 40 transgenic petunia plants derived from 26 transformed calli. Multiple shoots were regenerated from 8 of these calli and in only 6 cases were multiple regenerated shoots from each callus genotypically identical to each other. Many genotypes showed no nos gene expression (22/28). Most of the plants (16/22) which lacked nos gene expression did contain nos-encoding DNA with the expected restriction enzyme map. Similarly, amongst the genotypes showing no cab/ocs gene expression, the majority (11/28) did not show any alterations in restriction fragments corresponding to the expected cab/ocs coding sequences (10/11). Approximately half of the plants carried multiple copies of T-DNA in inverted repeats about the left or right T-DNA boundaries. No positive correlation was observed between the copy number of the introduced DNA and the level of expression of the introduced genes. However, plants with high copy number complex insertions composed of multiple inverted repeats in linear arrays usually showed low levels of expression of the introduced genes.