Intestinal N-acetylglucosamine 6-O-sulfotransferase (I-GlcNAc6ST, GST-4 alpha) and corneal N-acetylglucosamine 6-O-sulfotransferases (C-GlcNAc6ST, GST-4 beta) are two highly homologous GlcNAc 6-O-sulfotransferase isozymes encoded by two intronless open reading frames that reside similar to 50 kb apart on human chromosome 16q23.1. I-GlcNAc6ST has been shown to catalyze 6-O-sulfation of the endothelial mucin GlyCAM-1. C-GlcNAc6ST catalyzes 6-O-sulfation of GlcNAc in keratan sulfate and null-mutations in its encoding gene cause human macular corneal dystrophy. We show here that C-GlcNAc6ST efficiently catalyzes sulfation of GlyCAM-1 when coexpressed with the latter in COS-7 cells. We have further compared expression in human of both enzymes by Northern analysis with isozyme-specific probes. While I-GlcNAc6T is expressed mostly in intestinal tissue, larger C-GlcNAc6ST transcripts are found predominantly in the brain.