Various measures of biological activity were studied in batches of lyophilized Tice Bacillus Calmette-Guérin (BCG) that have been used intrapleurally as adjuvant therapy in surgically resected Stage I lung cancer patients by our own group, the North American Lung Cancer Study Group, and the Ludwig Lung Cancer Study Group. The biological activities of BCG that were studied were: (a) inhibition of solid tumor growth in mice following s.c. inoculation of BCG admixed with methylcholanthrene-induced fibrosarcoma cells (MC 43); (b) protection against tumor colonization of lungs and prolongation of survival in mice pretreated intrapleurally with BCG and later inoculated i.v. with a lethal dose of MC 43 tumor cells; and (c) growth properties in vitro, including a determination of the relative amounts of growing and nongrowing material and the sensitivity of growing material to inhibition by isoniazid. Significant differences in biological activity among batches of BCG were found. Some batches of BCG protected mice against circulating tumor cells, while others did not. Batches of BCG which protected mice against circulating tumor cells were found to have greater sensitivity to isoniazid and higher viability than batches which did not elicit this antitumor activity. There were also trends of some batches of BCG outperforming others in the clinic. Cumulative disease-free interval of patients was longer with batches which protected mice against circulating tumor cells than with batches which did not protect mice against circulating tumor cells. The results of this study suggest that preclinical testing of BCG for antitumor activity may improve the efficacy of this agent in future clinical trials.