A microspore-derived cell suspension culture of Brassica napus was used as a host for expression studies involving seed oleosin genes. The suspension culture was previously shown to display biochemistry and gene expression typical of zygotic embryos. Using a biolistic, transient expression approach we demonstrate that the seed-specific activator ABI3 promotes oleosin gene expression in these cultures. Co-bombardment of an oleosin promoter-GUS fusion and a full-length ABI3 gene from Arabidopsis resulted in four to six-fold enhancement of GUS expression. Deletion analysis was performed to identify which oleosin upstream sequences were required for ABI3 regulation. These studies found that a truncated oleosin promoter containing 160 bp of 5' regulatory sequence was sufficient to confer ABI3 responsiveness. Mutation of a canonical abscisic acid response element within this 160 bp region had a dramatic effect on basal expression, reducing levels to 25% of control. However, this mutation had no significant effect on ABI3 transactivation, indicating that the reduction in basal oleosin expression was distinct from the ABI3 response. These results also suggest that ABI3-mediated transactivation occurs through either a less-conserved ABRE element or other abscisic acid-independent sequences within the minimal promoter. Together, these data provide the first direct evidence that ABI3 mediates oleosin transactivation.