We have introduced the maize Ac transposable element in Arabidopsis thaliana and found that after three selfing generations, the element is immobile and extensively methylated. Moreover, the nopaline synthase (nos) gene present on the same transferred T-DNA, was active early after transformation and regeneration, but inactive in most of the S1 progeny. We used 5-azacytidine (5AzaC) to determine whether a reduction in the methylation would affect both Ac transposition and expression of the nos gene. After treatment with 5AzaC doses from 0.3 mM to 1.0 mM, approximately 25% of the plants produced detectable amounts of nopaline, indicating that the nos gene was reactivated. Using the polymerase chain reaction (PCR) to detect the empty donor site left by Ac transposition, we demonstrated that 5AzaC also activates Ac excision in the transgenic plants. Approximately 13% of the 5AzaC treated plants (doses from 0.1 mM to 1.0 mM) were shown to have empty donor sites due to Ac excision. None of the plants cultivated in the absence of 5AzaC showed evidence for Ac transposition or reactivation of the nos gene. Further analysis using Southern blot indicate that some demethylation occurred in the genome of individual plants. These results may represent demethylation in few cells during development which may be sufficient to reactivate in these cells the expression of the nos and Ac transposase transgenes, the latter promoting Ac transposition in somatic cells.