Three-day-old Penicillium charlesii mycelia were broken with Al2O3 in a buffered system and the membranes were separated on a linear gradient of sucrose concentrations. The most active guanosine 5'-(alpha-D-mannopyranosyl pyrophosphate):glycopeptide mannopyranosyltransferase (GPD-D-mannose mannosyltransferase) was found in two unresolved membrane fractions ( p congruent to 1.1 g/cm3). This preparation incorporated [14C]mannose from GDP-D-[14C]mannose both into endogenous acceptors and added peptidophosphogalactomannan. Mannosyltransferase activity is optimum at pH 7.0 in 0.05 M Tris/maleate buffer, and 17 mM Mn2+. Replacement of Mn2+ with Fe2+, Mg2, Co2+, Ca2+ or Ni2+ greatly reduced the mannosyltransferase activity. [14C]Mannose incorporation from GDP-D-[14C]mannose into acceptors is linearly dependent on enzyme concentration. Mannosyl incorporation into peptidophosphogalactomannan is linear for 3 h and continues for at least an additional 4 h. In contrast, the rate of mannosyl incorporation into endogenous acceptor(s) decreases after 60 min and there is no incorporation after 2 h. A series of possible acceptors related to peptidophosphogalactomannan were tested and it was found that treatment of peptidophosphogalactomannan with 0.01 N HCl at 100 degrees did not appreciably decrease the effectiveness of the acceptor even though this treatment removes the galactofuranosyl residues. In contrast, treatment of peptidophosphogalactomannan with 0.5 N NaOH rendered the products nearly incapable of accepting mannosyl residues from GDP-D-mannose. Derivation of peptidophosphogalactomannan with 2,4-dinitrobenzene also decreased its effectiveness as a mannosyl acceptor. [14C]Mannose from GDP-D-[14C]mannose was incorporated into both the oligosaccharide and phosphogalactomannan regions of peptidophosphogalactomannan. Treatment of the [14C]peptidophosphogalactomannan product, with 0.4 N NaOH released [14C]mannosyl-containing residues which eluted in the mannobiose and polysaccharide fractions from BIo-Gel P2. Approximately 90% of the 14C was in mannobiose. The [14C]mannose was shown to be transferred to the mannosyl-(seryl/threonyl) region of the acceptor. Acetolysis of [14C]peptidophosphogalactomannan resulted in the isolation of [14C]mannose, [14C]mannobiose, and [14C]polysaccharide. Small quantities of 14C were obtained in mannotriose and mannotetraose. The time course of [14C]mannose incorporation into the oligosaccharide region of peptidophosphogalactomannan showed a continual increase over a 4-h interval. In contrast, there was no major increase after 1 h in [14C]mannose incorporated into the polysaccharide region. The enzyme catalyzing incorporation of mannose into the polysaccharide region of peptidophosphogalactomannan was solubilized by treatment of the membranes with Triton X-100.