Pertussis toxin (PT), an AB5 exotoxin and important virulence factor of Bordetella pertussis, is hypothesized to traffic along a retrograde transport pathway in mammalian cells. This pathway includes endosomal uptake, transport to the Golgi complex and endoplasmic reticulum (ER), dissociation of the holotoxin in the ER and translocation of the A subunit (S1) to the cytosol, where it ADP-ribosylates its G protein targets. In this study, PT was visualized in the Golgi complex by immunofluorescence microscopy, but transport beyond the Golgi could not be detected by this method. To gain evidence for the retrograde pathway, peptide tags with target sites for tyrosine sulfation (a trans-Golgi network-specific activity) and N-glycosylation (an ER-specific activity) were added to either S1 or a B subunit (S4) of PT. Modified PT retained in vitro enzymatic and cellular activity as assessed by ADP-ribosylation assays. Peptide-tagged PT subunits were found to be modified by tyrosine sulfation, and, at later time points, by N-glycosylation. Appearance of sulfated PT subunits was inhibited by pretreatment of cells with brefeldin A. In some cell types, much of the S4 glycosylation, but not that of S1, was resistant to endoglycosidase H, suggesting that, subsequent to core N-glycosylation in the ER, S4 was transported anterograde to the Golgi, where further glycosylation occurred. When cells were pretreated with methyl-β-cyclodextrin, sulfation of PT subunits and PT cytotoxicity were reduced, suggesting that PT transport is dependent on cellular cholesterol content. These data support a retrograde pathway for PT intracellular transport.