Data on immune responses of young children using ATP release-based Cylex assay are insufficient. This study measured the immune response of healthy children less than three years of age to mitogens, PHA and Con-A. Blood was obtained from children attending routine health care visits. The Cylex assay was used to measure ATP production by CD4+ and CD3+ cells in response to PHA and Con-A, respectively. Samples from 20 children less than three years (range 10-27 months) were evaluated. The mean ATP production by CD4+ lymphocytes following PHA stimulation was 376 ng/mL (95% CI 17.1-735), which was similar to the response of older children in Hooper et al.'s (Clin Transplant 2005;19:834) study (p-value 0.28). The mean and median ATP production by CD3+ cells following Con-A stimulation were 114 ng/mL and 93.3 ng/mL, respectively (95% CI for median 45.2,148.6). The data suggest that although the immune system of young infants and toddlers is evolving, they are still able to respond to mitogen stimulation similar to older children.