A time resolved fluorometric immunoassay (TRFIA) has been developed and compared with an in house enzyme linked immunosorbent assay (ELISA) and commercial ELISA (Bindazyme) for the detection of tetanus antitoxin in human sera. A panel of 132 sera submitted for routine testing was used. Scatterplots showed a high degree of correlation between all three assays, although some divergence of results was apparent for low titre sera when comparing in house ELISA results with Bindazyme ELISA and TRFIA results. The TRFIA appeared to be more sensitive than the in house ELISA, and the Bindazyme assay compared well with the TRFIA. The intra-assay precision of all three assays, in terms of percentage coefficient of variation (%CV), was between 2.0% and 4.0%. The interassay precision ranged from 5% to 8% for the in house ELISA, 13% to 19% for the Bindazyme assay, and 11% to 13% for TRFIA. Both Bindazyme and TRFIA assays were simple to perform, accurate, reproducible, and amenable to automation. A particular benefit of the TRFIA was its large dynamic range, enabling tetanus antitoxin values of 0.01 IU/ml to 50 IU/ml to be measured with just one dilution of serum. TRFIA appears to be a useful serological technique worthy of further development.