Although the acute death syndrome in feline heartworm disease is widely recognized, its pathogenesis remains a mystery. The most widely held hypothesis is that an acute anaphylactic reaction, perhaps precipitated by the death of the parasite, is the underlying cause. This study investigated the role of the physical form of antigen (Ag) in the ensuing reaction when Dirofilaria immitis-sensitized cats are challenged by intravenous (IV) administration of heartworm Ag. Healthy D. immitis-naive cats (n = 23) were sensitized using subcutaneous injections of adjuvanted D. immitis Ag administered weekly for 6 weeks. After sensitization, cats (n = 20) were anaesthetized and challenged with IV D. immitis Ag in various forms or with IV sterile 0.9% saline (n = 3). Systolic blood pressure, respiratory rate, degree of dyspnea, blood oxygen saturation, and heart rate were measured immediately before and at 10-15 min intervals after challenge until terminal apnea occurred or until euthanasia at 140 min after challenge. Blood samples were collected for complete blood count and measurements of serum serotonin immediately before and at 10, 20, and 35 min after challenge. Clinical observations were recorded as they occurred, or at 10-15 minute intervals, whichever was the more frequent. The most severe post-challenge reactions occurred in cats challenged with Ag from dead worms, live worms, and 20 ng/mL Ag. Dyspnea increased significantly after challenge in all three groups (p < 0.001; p = 0.04, and p = 0.002, respectively), and blood oxygen saturation dropped post-challenge in the Dead Worm (p < 0.001) and the 20 ng/mL Ag (p = 0.002) groups. In the 20 ng/mL Ag group, systolic blood pressure decreased (p < 0.05) and respiratory rate increased (p < 0.05) post-challenge. Clinical observations included dyspnea, gastrointestinal signs (retching, defecation, or flatulence), urination, and less commonly, hemorrhage from the nostrils or anus, or cutaneous swelling (general or specifically facial). The 20 ng/mL Ag group had the highest rate of clinical signs, followed by the Dead Worm group. The most common and reliable hematologic change associated with severe clinical effects of D. immitis Ag challenge was increased hematocrit, which was statistically higher after challenge than at baseline in the Dead Worm group (p = 0.012). The model demonstrated that the physical form of heartworm Ag used for IV challenge in D. immitis-sensitized cats is an important factor for determining the characteristics of the post-challenge reaction, and the amount of exposed internal filarial Ag presented to the feline immune system may influence the severity of the response to challenge.