Radioimmunoassays specific for the N and C termini of human prothymosin alpha and the N terminus of human parathymosin alpha were employed for the measurement of the levels of alpha-thymosins in human thymus, spleen, and liver during normal growth and intestine and breast in malignant growth. A differential expression of the two alpha-thymosins was observed in thymus (prothymosin alpha-rich) and liver (parathymosin alpha-rich). A decline in the levels of both alpha-thymosins was found with age, with prothymosin alpha in thymus showing the sharpest change (15- to 30-fold). The levels of both alpha-thymosins were higher in malignant tissues as compared with healthy ones. In breast cancer, in particular, the mean increase for prothymosin alpha and parathymosin alpha was 17.9- and 11.5-fold, respectively. The major crossreactive material was characterized in all cases as intact prothymosin alpha and parathymosin alpha. These results suggest an in vivo relationship of the expression of alpha-thymosins with the human tissue cell proliferation activity.