In a previous transactivation screen, two Arabidopsis thaliana R2R3-MYB transcription factors, HAG2/MYB76 and HAG3/MYB29, along with the already characterized HAG1/MYB28, were identified as putative regulators of aliphatic glucosinolate biosynthesis. Molecular and biochemical characterization of HAG2/MYB76 and HAG3/MYB29 functions was performed using transformants with increased or repressed transcript levels. Real-time PCR assays, cotransformation assays and measurements of glucosinolate contents were used to assess the impact of both MYB factors on the steady-state level of glucosinolate biosynthetic genes and accumulation of aliphatic glucosinolates. Both HAG2/MYB76 and HAG3/MYB29 were shown to be positive regulators of aliphatic glucosinolate biosynthesis. Expression of promoter-beta-glucuronidase (GUS) fusions indicated GUS activities in both vegetative and generative organs, with distinct characteristics for each MYB factor. HAG1/MYB28, HAG2/MYB76 and HAG3/MYB29 reciprocally transactivated each other in the control of aliphatic glucosinolate biosynthesis and downregulated the expression of genes involved in the control of indolic glucosinolate biosynthesis, pointing to a reciprocal negative regulation of these two pathways. All three HAG transcription factors exert a coordinated control on aliphatic glucosinolate biosynthesis.