Regulation of IL-1 bioactivity by the IL-1 receptor antagonist (IL-1Ra) is critical for the preservation of normal vascular structure and function in mice. In humans, IL-1 bioactivity may be further fine-tuned by genetic polymorphisms. We recently proposed that an intronic polymorphism of the human gene encoding the IL-1R antagonist (IL1RN) is a genetic risk factor for the development of chronic cardiac allograft rejection. Here we aimed to establish a physiological basis for this susceptibility. Plasma and peripheral blood mononuclear cells (PBMC) were obtained from 55 healthy human volunteers, whose genotypes for four polymorphisms of IL1 family genes (IL1B, IL1R1 and IL1RN) were determined by PCR. IL-1beta and IL-1Ra production and release in PBMC cultures were studied by flow cytometry and ELISA. Functional and genotypic data were pooled and analyzed first by multivariate and, subsequently, by univariate statistical tests. We were able to confirm our observed association of IL1RN genotype with chronic cardiac allograft rejection using multivariate statistics. IL1RN genotype also emerged as the principal regulator of both constitutive and stimulated IL-1Ra and IL-1beta release. Allele IL1RN*2 was consistently associated with higher IL-1Ra and lower IL-1beta release, in a dosage-dependent manner. Conversely, IL1RN*2 carriage was associated with reduced production of the intracellular isoform of IL-1Ra. These genotypic effects were only observed with prolonged culture prior to stimulation and were not appreciably influenced by the presence of exogenous modulators of IL-1beta production. We conclude that IL1RN genotype is the principal determinant of IL-1beta bioactivity within theIL1 gene cluster in humans and discuss its putative role in disease.