An enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of tetanus antitoxin in human sera as an alternative to the toxin neutralization test in mice, the currently accepted method of assay. The ELISA was found to be simple and quick to perform and required only small amounts of materials. In addition, the assay was found to give reproducible estimates of antitoxin levels and to measure antitoxin at levels as low as 0.01 IU per ml, a sensitivity similar to that of the neutralization test. Furthermore, a comparison of the results of the ELISA and the neutralization test involving 80 human sera, including sera with both high and low antitoxin levels, showed close agreement in antitoxin levels obtained by the two methods. It was concluded that ELISA was an acceptable alternative to the toxin neutralization test in mice for the measurement of tetanus antitoxin levels in human sera.