Chemical analysis (high-performance liquid chromatography) and bioassay demonstrated the presence of compounds that seem to be components of the Ixodes scapularis arrestment pheromone. Only two purines, guanine and xanthine, were found in acidified saline extracts made from cast skins after molting of fed nymphs, fed larvae, and fecal/excretory exudates deposited by unfed adults on substrates in their environment. The ratio of guanine to xanthine was 10.6:1 in an extract from the nymphal skins versus 0.95:1 in an extract from the larval skins. Guanine, xanthine, and traces of a third purine, tentatively identified as 8-azaguanine, were found in extracts made from filter paper strips or washings from glass vials contaminated with tick feces and excreta left by unfed adults. 8-azaguanine may be a product of microbial degradation of the other purines rather than a natural product from the ticks. Low concentrations of ammonia also were detected in saline extracts of excreta from feeding ticks. Hematin also was found in NH₄OH extracts of the black fecal/excretory exudates deposited by the unfed ticks. Hematin was tentatively identified by comparison of spectra with that of the authentic standard. Bioassays demonstrated a strong positive arrestment response to cast skins found to contain a mixture of guanine and xanthine and to black fecal/excretory exudates containing guanine, xanthine, the putative 8-azaguanine, and hematin. A Noldus video tracking system using a CCD video camera and Ethovision Pro tracking software showed statistically significant increases in the frequency of visits to the treated zone versus the control. Ticks were significantly more likely to assemble in response to the tick exudates within as little as 3 h compared with the controls. Previous bioassay studies also showed strong positive responses to guanine, xanthine, other purines, and hematin. Comparisons with the arrestment pheromones of other tick species are described. The inclusion of the pheromone components in a permethrin-impregnated oily matrix, Last Call, increased the lethal activity of the product to 95% compared with only 65% in the formulation with permethrin alone. More detailed knowledge of I. scapularis arrestment pheromone may be useful for improving the efficacy of this tick-killing technology even further.