DNA synthesis in guinea pig thymocytes suspended in RPMI 1640 medium increased to a peak after 4-5 h in culture and was followed by increased mitotic activity, indicating that many thymocytes in S phase proceeded through G2 into mitosis. Addition of L-alanine to the medium markedly increased the DNA synthesis within 1 h and the mitotic frequency from 6 h. The increase in DNA synthesis when L-alanine was present in the medium was thus caused by an increased number of cells in S phase. Human thymocytes cultured in RPMI 1640 for 18 h had a low mitotic frequency. Addition of L-alanine immediately started DNA synthesis in the arrested thymocytes resulting in increased mitotic activity from 6 h later. The results show that L-alanine is a growth factor for guinea pig and human thymocytes and should be included in tissue culture media used for such cells. Growth of thymocytes in vitro was partly synchronized, and the mitotic studies indicated that many cells had entered S phase near the start of incubation.