The drug sensitivity of retinoblastoma cells obtained from 14 fresh primary materials(13 from enucleation and 1 from autopsy) and 2 cultured lines (Y-79 and WERI-Rb1) was determined using the human tumor clonogenic assay developed by Hamburger and Salmon. Components of the conventional soft agar medium were slightly modified to make them suitable for growing primary retinoblastoma cells. More than 5 colonies were formed by all 14 primary samples tested from the 500•~ 10• ôUH• ô3• ôUS• ôcells plated. More than 30 colonies per dish were formed from the 13 samples, with a median plating efficiency of 0.033%(0.005-0.400), and these were used in the• ôNH• ôin vitro• ôNS• ô measure mentsof drug chemosensitivities. They showed homogeneous sensitivity to the representative alkylating agent L-phenylalanine mustard; 13 out of 14 showed a decrease in the colony formation of more than 70%. The other drugs which were effective(more than 70% colony inhibition) against the primary retinoblastoma cells were: doxorubicin(7 out of 13), mitomycin C (7 out of 13), actinomycin D (4 out of 13),• ôNH• ôcis• ôNS• ô-diamminedichloroplatinum (II)(3 out of 13), nimustine(1 out of 13), and peplomycin(1 out of 13). Vincristine, bleomycin, 5-fluorouracil, methotrexate, dacarbazine, and cytosine arabinoside were not effective. When the chemosensitivity of retinoblastoma cells of the two established cell lines was examined by the same method, only L-phenylalanine mustard was effective against Y-79, and no drug was effective against WERI-Rb1.