An improved microELISA to measure thymosin alpha 1 (T alpha 1) is described which uses a rabbit antibody against T alpha 1 that has been absorbed with a synthetic C-14 fragment of T alpha 1. This assay is compared to the previous assay which used the whole antisera. The antibodies to T alpha 1 are preincubated with the standard or human sera overnight at 4 degrees C, then incubated for an additional 24 h in microtiter plates coated with T alpha 1. Using the whole antiserum, the average T alpha 1 level was 2480 +/- 1110 (mean +/- S.D.) pg/ml by ELISA and 2360 +/- 870 pg/ml by radioimmunoassay (RIA) in eight different samples of human cord sera. Using the N-specific absorbed antiserum the mean T alpha 1 level was 11,800 +/- 4800 pg/ml by ELISA and 10,600 +/- 5200 pg/ml by RIA. Recoveries of exogenously added T alpha 1 are complete (109 +/- 25% for whole and 108 +/- 15% for absorbed antisera). The absorbed antiserum has an increased affinity for the amino acid terminal region of T alpha 1 and the T alpha 1 values by use of absorbed antisera are significantly higher (3-5 x) than those measured using the whole antisera. Thus, the absorbed antisera produces an ELISA which is more sensitive and specific for serum thymosin alpha 1.