To investigate the role of individual Jun proteins in cell growth and transformation, we have used a doxycycline-inducible retroviral vector to regulate their expression in rat fibroblasts. AP-1 complexes enriched with cJun and JunB result in morphological alterations and anchorage-independent cell growth consistent with a transformation-like phenotype, whereas complexes enriched with JunD had an antiproliferative effect. These results suggest that genes regulated by both cJun and JunB are potentially involved in transformation and that they can be distinguished from those regulated by AP-1 complexes containing JunD. To identify genes regulated by cJun and JunB that may have a role in anchorage-independent growth, we investigated differential gene expression by each of the Jun family members using the Affymetrix Rat oligonucleotide microarray, RG_U34A containing approximately 8000 genes. Differentially regulated genes were identified and grouped for correlation with regulation by the different Jun proteins. A total of 33 candidate genes were found to be differentially regulated by both cJun and JunB and not by JunD. These genes have roles in cell metabolism, growth, signal transduction, migration and adhesion. We validated the differential regulation by cJun and JunB of 10 candidate genes by Northern blot analysis. Of these, eight were further characterized as potential direct targets of AP-1 regulation based on Northern blot results showing differential regulation that correlate with cJun expression. Our results show that inducible cJun and JunB expression result in anchorage-independent growth of Rat1a cells, distinct from JunD-expressing cells. This model system and a functional genomic approach enabled us to differentiate AP-1-regulated genes involved in transformation from AP-1-regulated genes known as bystander genes. This approach significantly reduces the number of bystanders and allows for the targeting of genes specifically involved in transformation.