Background: Few studies have characterized the clinical manifestations of delayed antibiotic hypersensitivity (AH) diagnosed using objective methods. The lymphocyte transformation test (LTT) is a reproducible method to diagnose type IV hypersensitivity. The purpose of the study was to evaluate the characteristics of delayed AH diagnosed on LTT in children. Methods: We performed a retrospective analysis of patients who were evaluated for AH using LTT at National Center for Child Health and Development, Tokyo, from 2002 to 2014. We extracted patient demographics, type and duration of antibiotics, and clinical characteristics from the medical records. Clinical manifestations were compared between LTT-positive and LTT-negative cases. Results: Seventy-five cases for which 101 drugs were tested were included in this study. LTT was positive against 34 drugs in 26 cases. Median age was 5 years (IQR, 1-9 years), and 49% of patients had underlying disease. LTT was performed at a median of 18 days (IQR, 4-59 days) after the suspected episode. The median number of days from the initiation of therapy to the onset of symptoms was 4. Rash was the most common manifestation (89%). Fever (>38°C) was observed in 20 cases (27%). Onset of fever preceded the rash in nine cases (45%), appeared simultaneously in five (25%), appeared afterwards in four (20%), and was unknown in two (10%). Fever was an independent factor associated with AH when comparing LTT-positive and LTT-negative cases (OR, 3.61; 95%CI: 1.03-12.64). Conclusions: Fever was a common presenting symptom of delayed AH in children aged ≤18 years.