Background α and β-Tryptase levels in serum are clinical tools for the evaluation of systemic anaphylaxis and systemic mastocytosis. Basophils and mast cells are known to produce these proteins. Objective The current study examines the effect of the α,β-tryptase genotype on basophil tryptase levels and the type of tryptase stored in these cells. Methods Tryptase extracted from purified peripheral blood basophils from 20 subjects was examined by using ELISAs measuring mature and total tryptase and by using an enzymatic assay with tosyl-Gly-Pro-Lys-p-nitroanilide. Tryptase genotypes (4:0, 3:1, and 2:2 β/α ratios) were assessed by using a hot-stop PCR technique with α,β-tryptase–specific primers. Total α,β-tryptase mRNA was measured by means of competitive RT-PCR, and ratios of α to β-tryptase mRNA were measured by means of hot-stop RT-PCR. Results Tryptase in all but one of the basophil preparations was mature and enzymatically active. Tryptase quantities in basophils were less than 1% of those in tissue mast cells. Tryptase genotypes (β/α) among the 20 donors were 4:0 in 7, 3:1 in 7, and 2:2 in 6. Tryptase protein and mRNA levels per basophil were not affected by the tryptase genotype. Conclusion Basophils from healthy subjects contain modest amounts of mature and enzymatically active tryptase unaffected by the tryptase genotype.