Tryptase, a tetrameric serine protease, is a main constituent of the secretory granules in human mast cells, where it is stored in complex with heparin or chondroitin sulfate proteoglycan. Human tryptase has been implicated in a variety of clinical conditions including asthma, but the mechanisms that lead to its tetramerization/activation have not been extensively investigated. Here we addressed the activation mechanisms for human betaI and betaII-tryptase, which differ in that betaI-tryptase is N-glycosylated at Asn102 whereas betaII-tryptase has a Lys residue at position 102, and consequently lacks the corresponding N-glycosylation. We found that both tryptases were dependent on heparin for activation/tetramerization, but whereas betaI-tryptase activation preferentially occurred at acidic pH, betaII-tryptase activation was less pH-dependent. Both betaI and betaII-tryptase bound strongly to heparin-Sepharose at acidic pH but with lower affinity at neutral pH. Further, while addition of heparin to betaI-tryptase predominantly resulted in formation of active tetrameric enzyme, betaII-tryptase showed a tendency to form inactive aggregates. betaI and betaII-tryptase were similar in that the minimal heparin size to induce activation was an octasaccharide and in that the interaction with heparin and structurally related polysaccharides was dependent on high anionic charge density rather than on specific structural motifs. Addition of decasaccharides to both betaI and betaII-tryptase resulted in the formation of active monomeric enzyme, whereas intact heparin promoted assembly of tetrameric enzyme. This, together with a bell-shaped dose response curve for heparin-induced activation, suggests that the mechanism for tetramerization involves bridging of individual tryptase monomers by heparin. Taken together, this study indicates a key role for heparin in the activation of human beta-tryptase.