A panel of 60 human tumor cell lines is currently being used in the U.S. National Cancer Institute's in vitro anticancer drug screen. The panel is organized into 7 subpanels; 6 leukemia/lymphoma lines comprise one subpanel, and 54 other lines are organized into subpanels representing solid tumors of the central nervous system (CNS), colon, lung, ovaries, kidneys and melanomas. In the present study, the leukemia and lymphoma cell lines were analyzed by flow cytometry for appropriate CD antigens; all but 1 line showed patterns of expression consistent with their reported derivations. The solid tumor lines were characterized individually using morphological and immunocytochemical techniques to determine their relative degrees of representativity for the subpanels within which they are currently grouped. Histological, histochemical and ultrastructural examinations were performed on cell lines grown under identical conventional culture conditions and as xenografts in nude mice. Immunocytochemistry using panels of antibodies raised against 6 types of intermediate filaments, 7 adenocarcinoma-associated antigens, 7 melanoma/neuro-ectodermal-associated antigens, 3 neuroendocrine-associated antigens, 9 urinary tract associated antigens, and 4 markers of muscle differentiation was done on cells grown in monolayer culture. Central nervous system (CNS) cell lines lacked expression of glial fibrillary acidic protein, but all had other features consistent with derivation from glioblastoma. Lines derived from adenocarcinomas of the colon, lung and ovary, for the most part, expressed adenocarcinoma-associated antigens and showed histological and/or ultrastructural evidence of gland formation and other adenomatous features. Most of these lines were poorly differentiated. Lines derived from large-cell and squamous-cell cancers also showed some characteristics consistent with their reported origins, except for one line which showed immunocytochemical and morphologic characteristics consistent with rhabdomyosarcoma. The 2 lines derived from small cell lung cancer (SCLC) lacked neurosecretory granules and 3 other SCLC markers but showed morphologic features consistent with SCLC. Most melanoma cell lines strongly expressed melanoma-associated antigens and were morphologically similar to human melanoma. Five lines produced premelanosomes, melanosomes or melanin. Most of the renal cancer cell lines showed morphologic or immunocytochemical features consistent with renal clear cell carcinoma. Collectively, these morphological and immunocytochemical analyses provide information concerning tissue of origin, tumor type, degree of differentiation and other biologic features essential to the use of these lines in a disease-oriented in vitro antitumor drug screen and to the interpretation of data derived therefrom.