Background: Café-au-lait macules (CALMs) in neurofibromatosis type 1 (NF1) are an early and accessible phenotype in NF1, but have not been extensively studied. Objective: We sought to more fully characterize the phenotype of CALMs in patients with NF1. Methods: In all, 24 patients with a diagnosis of NF1 confirmed through clinical diagnosis or molecular genetic testing were recruited from patients seen in the genetics department at the University of Alabama at Birmingham. CALM locations were mapped using standard digital photography. Pigment intensity was measured with a narrowband spectrophotometer, which estimates the relative amount of melanin based on its absorption of visible light. The major response was defined as the difference between the mean melanin from the CALM and the mean melanin from the surrounding skin. The major response for each spot was compared with spots within an individual and across individuals in the study population. Results: There was significant variability of the major response, primarily attributable to intrapersonal variability (48.4%, P < .0001) and secondly to interpersonal variability (33.0%, P < .0094). Subsequent analysis based on genetic mutation type showed significantly darker spots in individuals with germline mutations leading to haploinsufficiency. Limitations: The study was performed on a small population of patients and the method has not yet been used extensively for this purpose. Conclusions: CALMs vary in pigment intensity not only across individuals, but also within individuals and this variability was unrelated to sun exposure. Further studies may help elucidate the molecular basis of this finding, leading to an increased understanding of the pathogenesis of CALMs in NF1. © 2009 by the American Academy of Dermatology, Inc.