Tumor infiltrating lymphocytes (TIL) from malignant ascites or solid tumor specimens obtained from patients with ovarian carcinoma were expanded to large numbers in vitro (10(10)-10(11)) by a four-step method using AIM V medium and low concentrations of recombinant interleukin-2 (rIL-2). The expansion procedure employed 24-well culture plates, T-flasks, polyolefin gas-permeable bags (PGPB), and an artificial capillary culture system (ACCS). The mean number of mononuclear leukocytes introduced into the 24-well plates was 16.5 +/- 4.2 x 10(6) cells. TIL from a total of 16 patients were expanded only through the first three steps of the process (24-well-plates, T-flasks, and PGPB) with an overall expansion of 255 +/- 99 fold and mean duration of 27.4 +/- 2.2 days. TIL from 9 of 16 patients were expanded further through the fourth step (ACCS) of the expansion method. The cumulative fold-expansion in nine patients was 8044 +/- 4807 (mean +/- SEM), the median was 2876 and the mean expansion time was 47.1 +/- 4.7 days. TIL from seven additional patients did not grow in rIL-2. Six of these 7 patients received chemotherapy at least four weeks before the specimens were collected. Two ACCS were used in parallel to facilitate expansion of TIL. Viable rIL-2-expanded TIL in the range of 1 x 10(10)-1 x 10(11) were recovered from the two ACCS, a number sufficient for adoptive immunotherapy of patients with ovarian carcinoma. The rIL-2-expanded TIL were predominantly CD3+ CD4+ CD8- alpha beta TCR+, although CD3+ CD4- CD8+ alpha beta TCR+ T cell lines were obtained from certain patients. An increase (43 +/- 8 vs 75 +/- 13; P = 0.05) in the proportion of CD4+ cells was observed over the duration of the four expansion steps. However, CD8+ TIL-derived T cells lines were also expanded in the ACCS. The four-step expansion method described here has several significant advantages over existing techniques. It requires substantially less personnel, equipment and space and the risk of contamination during expansion of the cultures is decreased. These results demonstrate that the four-step method described here can be effectively used for the large-scale expansion of ovarian TIL for the treatment of patients with ovarian carcinoma by adoptive immunotherapy.