Yeast Saccharomyces cerevisiae OCH1 gene encodes the mannosyltransferase that is essential for the outer chain elongation of N-linked oligosaccharides. Mannosyltransferase activity of OCH1 gene product (Och1p) was measured on HPLC by using pyridylaminated Man8GlcNAc2 (Man8GlcNAc2-PA) as an acceptor and the reaction product was observed at the retention time corresponding to Man9GlcNAc2-PA. 1H-NMR and fast atom bombardment mass spectrometry (FAB-MS) fragmentation analysis of Man9GlcNAc2-PA showed that the additional mannose was attached with an alpha-1,6 linkage at the site where mannose outer chain elongation initiates. Substrate specificity of Och1p was investigated by using various high mannose-type oligosaccharides as acceptors. Man8GlcNAc2 was the best acceptor for Och1p. The loss of one or two alpha-1,2-mannoses from Man8GlcNAc2 reduced the mannosyltransferase activity and the Man5GlcNAc2 completely lacking alpha-1,2-mannose residues did not serve as an acceptor. Man8GlcNAcOH that involves an open sugar ring by reduction of reducing terminal GlcNAc residue did not serve as an acceptor for Och1p. The loss of three mannoses at the alpha-1,6-branch also reduced the Och1p activity. These results suggest that Och1p is an initiation specific alpha-1,6-mannosyltransferase that requires the intact structure of Man8GlcNAc for efficient mannose outer chain initiation.