Azo dyes form a major class of chemically related compounds that are ubiquitous in foods, paints, printing inks, cosmetics, and also used as biological stains in histological and histopathological laboratories and clinics. Sudan I, sudan III, and sudan IV have been classified as category 3 carcinogens by International Agency for Research on Cancer. In this study, we investigated the difference between these three sudan dyes in induction of CYP1A1. We intraperitoneally treated Wistar rats with each of the three sudan dyes (I, III, and IV) for 3 days. Treatment of Wistar rats with sudan I produced the highest induction of CYP1A1 protein and mRNA whereas treatment of Wistar rats with sudan III produced about two third of CYP1A1 protein and mRNA than induced by sudan I. Furthermore, treatment of Wistar rats with sudan IV produced the lowest induction of CYP1A1 protein and mRNA which is about two third of that induced with sudan III treatment. We further investigated the effect of these sudan dyes on CYP1A1 transcription through investigating the xenobiotic response element (XRE) reporter activity in HepG2. The XRE reporter activity study showed the same trend of activity of sudan dyes comparable to the effects on CYP1A1 mRNA and protein. Immunohistochemical study revealed a differential pattern of distribution of CYP1A1 protein in rat liver among the three sudan dyes, apparent in the centrilobular and midzonal region with sudan III, progressing to panlobular with sudan I, whereas sudan IV showed a reversal of pattern of induction with the most intense staining in the periportal region. Our results suggest that there is an inverse relationship between the molecular size of the three sudan dyes and their ability to induce CYP1A1.