Biotinidase, the enzyme responsible for recycling the vitamin biotin, is deficient in most individuals with late-onset multiple carboxylase deficiency. Based on clinical criteria, biotinidase deficiency appears to be inherited as an autosomal recessive trait; however, the inheritance of biotinidase serum activity as a quantitative trait has not been studied previously. In this study, both segregation analysis of proband families and the analysis of twin family data were used to determine the relative contributions of a major gene, polygenes and environment to the variation in serum biotinidase activity. Segregation analysis of 24 families of biotinidase-deficient individuals indicated that serum biotinidase activity is determined by the segregation of a single codominant major gene with the variability about the mean of each major genotype attributable to environmental effects. Significant polygenic effects could not be detected by this analysis. Variance component analysis of 128 twin families, which included the twins, their spouses, and their offspring, indicated that 70% of total variance in biotinidase activity is attributable to additive genetic effects, 22% to individual environmental effects, and 8% to shared environmental effects. The model also included an age effect for females. A portion (27%) of the estimated additive variance may be attributed to the segregation of the major gene. This study emphasizes the usefulness of studying multiple data sets representing different types of family relationships.