OBJECTIVE: The risk of infantile hypertrophk pylonc stenosis is ∼5 times more common in male than female infants. It has been hypothesized that the higher risk among male infants is associated with high levels of testosterone causing hypertrophy of the pylorus muscle. To test this hypothesis, we examined the association between the testosterone levels in the umbilical-cord blood and the risk of infantile hypertrophic pyloric stenosis. PATIENTS AND METHODS: We conducted a matched case-control study nested in the Danish National Birth Cohort using risk-set sampling. From a cohort of 101 042 pregnancies, we identified umbilical-cord blood samples from 46 case subjects (43 male and 3 female infants) who developed infantile hypertrophic pyloric stenosis in the first year of life and 150 gender-and gestational age-matched control subjects. The testosterone levels were measured by liquid chromatography-tandem mass spectrometry. Rate ratios were estimated by using conditional logistic regression. RESULTS: In male infants, the mean testosterone level at birth was 0.78 nmol/L in case subjects and 0.91 nmol/L in control subjects. The rate of infantile hypertrophic pyloric stenosis was inversely, albeit insignificantly, associated with the testosterone levels in male infants; there was a 29% (95% confidence interval: -46% to 65%; P = 35) lower rate per nmol/L. The association was not modified according to age, gestational age, or birth order. CONCLUSIONS: We found no support for the hypothesis that high testosterone levels in the umbilical-cord blood are strongly associated with a subsequently higher risk for infantile hypertrophic pyloric stenosis in male infants.