A recombinant expression system was developed to analyse sequence determinants involved in O-glycosylation of proteins in mycobacteria. By expressing peptide sequences corresponding to known glycosylation sites within a chimeric lipoprotein construct, amino acids flanking modified threonine residues were found to have an important influence on glycosylation. The expression system was used to screen mycobacterial sequences selected using a neural network (NetOglyc) trained on eukaryotic O-glycoproteins. Evidence of glycosylation was obtained for eight of 11 proteins tested. The results suggest that sites involved in O-glycosylation of mycobacterial and eukaryotic proteins share similar structural features.