Current methods of expanding tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma bulk cultures result in a heterogeneous population of cells with low tumor-killing specificity. To improve the yield of cells with higher autologous and lower nonspecific cytotoxicity, interleukin-4 (IL-4) was added to high (1,000 U/ml)- and low (20 U/ml)-dose IL-2 and compared to cultures grown without IL-4 for proliferation, phenotype, and cytotoxicity against targets including autologous and allogeneic tumors. When compared to culture in IL-2 alone, the addition of IL-4 improved overall expansion in both high-dose (mean fold expansion of 2,061 vs. 1,087) and low-dose (mean fold expansion of 1,904 vs. 262) IL-2. Enhancement of TIL proliferation was dependent on the timing of IL-4 addition to the culture; augmented growth occurred only when IL-4 was added with or following activation by IL-2. The phenotype consisted primarily of CD3+/CD4+ lymphocytes with a reciprocal reduction in CD56+/CD16+ cells. Finally, there was a significant reduction in nonspecific cytotoxicity against K-562, M-14, and allogeneic tumor targets, but no significant change against autologous tumor. We conclude that IL-4 has an important regulatory effect on the expansion of renal cell carcinoma TILs in IL-2 by the promoting growth of CD3+/CD4+ lymphocytes and inhibiting the growth and nonspecific cytotoxicity associated with LAK-like CD16+/CD56+ cells. These findings may be beneficial in extracting more potent effector cells from bulk TIL culture for use in clinical trials.