Acquired delayed-onset hypolactasia is a common autosomal recessive condition. Cow's milk allergies, conversely, are less common conditions that may manifest with equivalent symptoms and are able to simulate and/or aggravate lactose intolerance. This study was designed to evaluate the contribution of IgE-mediated cow's milk sensitization to the symptomatology of adult patients with lactose-free diet refractory lactose intolerance. Forty-six adult patients with lactose intolerance and persistent symptoms despite a lactose-free diet underwent skin-prick test to investigate cow's milk, goat's milk, and soy protein-specific-IgE. SDS-PAGE immunoblotting was used to investigate the presence of cow's milk protein-specific IgE. The percentage of patients who had skin reactions to whole cow's milk, alpha-lactalbumin, beta-lactoglobulin, caseins, goat's milk, and soy was 69.5, 36.9, 56.5, 56.5%, 54.3, and 50%, respectively. The percentage of patients with immunoblot-detected IgE specific for alpha-lactalbumin, beta-lactoglobulin, caseins, and bovine serum albumin was 21.7, 63, 67.3, and 2.1%, respectively. IgE-mediated sensitization to cow's milk is a frequent comorbidity in subjects with lactose-free diet refractory lactose intolerance and is worth consideration in patients with this condition.