BACKGROUND: Pharmacological stress is often used, and the drugs most frequently utilized are dipyridamole (Dip) and dobutamine (Dob). We aimed to evaluate the safety and the results obtained with a new protocol associating Dip, Dob, and atropine, compared with the Dip protocol. METHODS: Thirty-two patients underwent rest Tl/Dip stress Tc-sestamibi myocardial perfusion tomography on the same day. Dip was administered intravenously (0.56 mg/kg) for 4 min, and Tc-sestamibi was injected 3 min after the end of the Dip injection. On another day, patients received the same Dip dose, immediately followed by the infusion of Dob [20 µg/kg/min for the first 2 min and 40 µg/kg/min in the next 2 min, with atropine (1 mg) given in the interval between the two Dob doses]. Images were acquired with a two-detector camera. In a 17-segment model of the left ventricle, each segment was automatically scored 0-4 (normal to absent radiotracer activity), and perfusion scores were obtained as the sum of stress score and sum of rest segmental score (SSS and SRS, respectively) and the difference between them [summed difference score (SDS)]. All scans were interpreted by two experienced physicians as either definitely normal, probably normal, probably abnormal, or definitely abnormal. RESULTS: No serious complication was found independently of the protocol used. Heart rate and systolic and diastolic blood pressure were similar in both protocols at the basal level. Maximum heart rate (126 ± 21 vs. 82.7 ± 13.6, P<0.001) and the double product (18816 ± 4194 vs. 11449 ± 2438, P<0.001) showed a significant increase in the tests that used Dob and atropine compared with the Dip protocol. Stress studies with Dip-Dob had higher SSS compared with the Dip protocol (9.4 ± 10.1 vs. 7.7 ± 8.8, P<0.001). SDS was also higher in the Dip-Dob protocol than in the Dip protocol (6.1 ± 6.8 and 4.8 ± 5.8, respectively, P< 0.001). CONCLUSION: This work shows that a new protocol of pharmacological stress with a combination of Dip, Dob, and atropine is safe, easy to administer, and results in larger stress-induced defect size and reversibility of myocardial perfusion.