Background: Concerns for sensitization after penicillin skin testing are a factor in limiting the timing and population for whom this testing is offered. The sensitizing potential of the penicillin skin test has never been studied directly. Methods: A total of 329 volunteers underwent prick and intradermal skin testing with penicillin G, benzylpenicilloylpolylysine, and a minor determinant mixture. Those with negative skin testing had repeat testing 4 weeks later. Medical history and antibiotic use were determined by interview, questionnaire, and electronic pharmacy records. Results: Seventy-two of the 329 subjects (22%) reported a history of previous β-lactam reaction, of which 10 (14%) had a positive initial skin test. Overall, the initial skin test was positive in 23 of 329 (7%). Of the subjects with a negative initial skin test, 239 completed the second test 4 weeks later. Of these, 6 subjects (2.5%, 95% confidence interval 0.5% to 4.5%) converted to a positive skin test. None had taken a β-lactam antibiotic between the two tests, and none had any previous history of β-lactam reaction. One subject reported having never taken a β-lactam antibiotic before. In comparison to the 233 subjects who did not convert their skin test, the statistically significant factors favoring sensitization were: female sex (odds ratio [OR] 6.53, P = 0.05), atopy (OR 5.31, P = 0.04), and history of food allergy (OR 6.35, P = 0.02). There was a trend toward more recent penicillin use in the newly sensitized subjects, but this was not statistically significant. Conclusion: Penicillin skin testing may sensitize a small number of individuals to penicillin.