A monoclonal antibody to the non-thymosin alpha 1 overlapping sequence of parathymosin alpha was used to develop a micro ELISA to measure parathymosin alpha. Parathymosin alpha levels were measurable in both mouse tissue extracts and in human adult and cord serum. To measure parathymosin alpha, a 40% ammonium sulfate precipitated monoclonal antibody specific for the parathymosin alpha fragment was preincubated with the parathymosin alpha fragment or samples for 4 hours at 4 degrees C, incubated an additional 24 hours in microtiter plates coated with the parathymosin alpha fragment and then assayed by the biotin-avidin-alkaline phosphatase method. Using the assay, parathymosin alpha could be measured over a range of 5 to 630 ng/ml and cross-reactivity with thymosin alpha 1 was not observed. The parathymosin alpha level was 400 +/- 140 (mean +/- SD) ng/ml in normal human sera (n = 12) and 150 +/- 80 ng/ml in cord sera (n = 12). The concentration of parathymosin alpha was highest in tissue extracts of mouse liver (153 +/- 40 micrograms parathymosin alpha per g of tissue), kidney (125 +/- 33), and lung (105 +/- 38). Levels were lowest in thymus (81 +/- 20), spleen (83 +/- 23), and brain (71 +/- 23).