Human malignant pleural mesothelioma (HMPM) is an aggressive neoplasm that is highly resistant to conventional therapies. We established 3 HMPM cell lines (TCC-MESO-1, TCC-MESO-2 and TCC-MESO-3) from Japanese patients; the first 2 from the primary and metastatic tumors of a patient with the epithelioid type of HMPM, and the third from a patient with biphasic characteristics of the tumor (epithelioid and sarcomatous phenotypes). The 3 cell lines resembled the original HMPMs in their morphological and biological features, including the genetic alterations such as lack of p16 expression and mutation of p53. Their tumorigenicity was determined in SCID mice by orthotopic implantation (20-46%). The tumorigenicity of the HMPM cell lines, which was relatively low, was enhanced by repeated subcultures and orthotopic implantations, and 3 competent tumorigenic sublines were produced (Me1Tu, Me2Tu and Me3Tu sublines from the TCC-MESO-1, TCC-MESO-2 and TCC-MESO-3 cell lines, respectively). The resultant HMPM sublines efficiently generated tumors in the SCID mice (100%) following orthotopic implantation. SCID mice implanted with the competent sublines, into one of which the luciferase gene was introduced, displayed quantitative fluctuation of the bioluminescence for the tumor volume in vivo. Oral administration of S-1, an anticancer agent, suppressed the proliferation of the luciferase gene-expressing Me1Tu subline in the mouse models in vivo, with a treated-to-control ratio of the mean tumor volume of 0.2. The orthotopic implantation mouse model proved to be useful for quantitative evaluation of the efficacy of novel anticancer drugs and also for studying the biology of HMPMs in vivo.