ortho-Phthalaldehyde (OPA) has been used as a safe alternative disinfectant instead of glutaraldehyde; however, recently some adverse effects of OPA were reported in patients and medical professions. We examined the acute toxicity of OPA in male ICR mice injected with 0.125-0.5% OPA and killed some animals 1 day after a single OPA injection, and others 1 or 13 days after two OPA injections 5 days apart. Hematology, blood cell counts, specific antibody production, organ weights, hepatic enzymes, hepatic histopathology and gene expression of cytochrome P450 (CYP) mRNA in liver were examined. Single OPA injections elevated leukocyte counts, the proportion of neutrophils, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Two OPA injections dose-dependently increased leukocyte counts, the proportion of neutrophils, ALT and AST, and decreased alkaline phosphatase. Leukocyte counts and proportions of neutrophils normalized 13 days after the second of two injections. However, both ALT and AST remained high in mice given higher OPA doses. Significant increased liver-to-body weight ratio and mild hepatic lesions were observed. Gene expression of CYP1a1 and CYP2e1 revealed a tendency of up-regulation 1 day after two OPA injections. However, expression of these genes was then down-regulated 13 days after OPA injections. OPA induced specific IgE and IgG significantly in the sera, suggesting that OPA acts as a hapten. Overall, OPA caused acute inflammation and acted as a haptenic allergen, although it caused only mild liver injury. Such evidence suggested that careful washing and prevention of exposure were needed after OPA disinfection of medical instruments.