Tryptase (alpha and beta) levels in serum are used to assess mast cell involvement in human disease. Using cultured cells, the current study examines the hypothesis that protryptase(s) are spontaneously secreted by mast cells at rest, whereas mature tryptase(s) are stored in secretory granules until their release by activated cells. HMC-1 cells have only beta-tryptase genes and the corresponding mRNA. Mono-Mac-6 cells have both alpha- and beta-tryptase genes but preferentially express alpha-tryptase. Mono-Mac-6 cells spontaneously secrete most of their tryptase, which consists of alpha-protryptase, whereas mature tryptase is retained inside these cells. HMC-1 cells also spontaneously secrete most of their tryptase, identified as beta-protryptase, and retain mature tryptase. Skin-derived mast cells retain most of their tryptase, which is mature, and spontaneously secrete protryptase(s). Total tryptase levels in plasma are detectable but no different in healthy subjects with and without the gene for alpha-tryptase, consistent with pro forms of both alpha- and beta-tryptase being spontaneously secreted. Thus, protryptase(s) are spontaneously secreted by resting mast cells, whereas mature tryptase is retained by mast cells until they are activated to degranulate.