The current recommendation for safe and adequate daily dietary intake of biotin for infants is based on measurements of biotin concentration in human milk and calculations of biotin intake that tacitly assume that biotin content of human milk is reasonably uniform for a given subject. This assumption of uniformity was tested by examining the effects of several factors on the biotin concentration. The degree of breast emptying had little effect on biotin concentration. However, in three of five individuals studied, the biotin concentration varied significantly over 24 h. In two of five subjects, there was a consistent difference between breasts of approximately 16%. In the first 18 d postpartum, the milk concentration of biotin increased steadily in four of the eight individuals studied, remained low in two and increased erratically in two. Rather than reaching a stable plateau in mature milk, biotin concentration varied substantially in most of the subjects. These observations provide evidence that an adequate scheme for estimating total biotin intake of the breastfed infant will require sampling from both breasts frequently over the 24-h cycle and frequently as a function of time postpartum.