We measured anti-tick saliva antibody (ATSA) by enzyme-linked immunosorbent assay using whole sonicated Ixodes dammini salivary glands as antigen in subjects with 1) a recent and confirmed I. dammini (n = 100) or Dermacentor variabilis bite (n = 3), 2) erythema migrans (n = 15), 3) late-stage Lyme disease (n = 4), and 4) normal controls without a history of tick bites (n = 5). Tick bite subjects had three ATSA determinations over approximately six weeks. On the first ATSA measurement at a mean +/- SD of 18.5 +/- 19.8 hr after removal of the tick, the subjects bitten by I. dammini had a mean ATSA optical density value (95% confidence interval [CI]) of 0.264 (0.223, 0.305); the corresponding value in controls was 0.142 (0.115, 0.169). There was no consistent change in ATSA levels in individuals with time. Multiple linear regression indicated that tick engorgement (P < 0.01), subject age (higher ATSA with increasing age; P = 0.01), and subject sex (females > males; P = 0.03) were all independent predictors of ATSA levels. Logistic regression revealed that a bite by I. dammini that became engorged (defined as an engorgement index > or = 3.4) was a risk factor for ATSA seropositivity (odds ratio [95% CI] = 6.2 [1.7, 21.8]). Finally, the ATSA test had a sensitivity of 0.81 and a specificity of 0.56 for a bite by I. dammini that became engorged. Overall, the data are further evidence that ATSA is a biologic marker of tick exposure, in that the engorgement index, a surrogate for tick saliva dose, was the strongest independent predictor of antibody response.