The present study sought to determine the expression of α‐ and β‐tryptase in in vitro differentiated human cord blood derived mast cells. We also analysed the glycosaminoglycan composition and the phenotype of the cells. The major protease in human mast cells is tryptase, and cDNAs for two different human tryptases have been characterized, the so‐called α‐ and β‐tryptase. By reverse transcriptase‐polymerase chain reaction (RT‐PCR) we could show that stem cell factor (SCF)‐dependent cord blood derived mast cells express both α‐ and β‐tryptase. Furthermore, the cells were stained with a monoclonal antibody (mAb) against tryptase, and the tryptase was enzymatically active cleaving the substrate Z‐Gly‐Pro‐Arg‐ methoxy‐2‐naphthylamide (MNA). The majority of the cord blood derived mast cells could also be stained with mAbs against chymase, cathepsin G and CD68. They also expressed Kit/SCFR (CD117), CD13, CD29 and CD45 on the cell surface. The proteoglycan‐derived polysaccharide composition of the cells was estimated to be 25–35% of heparin origin and 65–75% of chondroitin sulphate origin. Hence, the cord blood derived mast cells exhibit a phenotype in common with the so‐called MC_TC type of human mast cells.