Up to one-third of haemophilia A patients develop factor VIII (FVIII) alloantibodies (inhibitors). The Bethesda assay detects inhibitors but is relatively insensitive. Recently, a new fluorescence-based immunoassay (FLI) was developed for antibody detection. The aim of this study was to assess the prevalence of inhibitors as measured by FLI. Assays of FVIII, FVIII inhibitor by Bethesda assay with Nijmegen modification, and FVIII inhibitor by FLI were performed on adult patients with haemophilia A. Data were complete for 46 patients (median age 39), of whom 72% were severe, 7% moderate and 22% mild. The Bethesda assay was positive in only two patients (4%), while FLI was positive in 23 of 46 patients (50%), with values ranging from 0.4 to 33.7 nm (median 3.5 nm). FLI titres exceeded 7.0 nm in 19.5% of patients, all but one of whom had severe haemophilia. FLI antibody-positive patients were less likely to be HIV positive (30% vs. 70%, P = 0.02). The use of a prophylaxis regimen was associated with a lower incidence of antibody; only two of 23 patients with detectable antibody and none of those with antibody >7 nm were on a prophylaxis regimen, while nine of 23 patients without antibody were on prophylaxis, (P = 0.03). There was no difference in inhibitor presence in patients using recombinant versus plasma-derived factor. Antibodies detected by FLI are frequent in patients with haemophilia A, but are less common in those who are HIV positive or are receiving regular FVIII prophylaxis.