When cardiologists diagnose patients with coronary spastic angina, Japanese Circulation Society (JCS) guidelines recommend the intracoronary injection of acetylcholine (ACh) and ergonovine (ER) as class I. However, the pharmacological difference between ACh and ER is controversial in the clinic. We performed both ACh and ER tests in the same 528 patients during 26 years. We investigated the provoked spasm configuration, spasm site, and clinical characteristics of provoked spasm between ACh and ER, retrospectively. We defined positive spasm as ≥90% luminal narrowing. Provoked positive spasm was observed in 161 right coronary arteries (RCA) including 83 ACh just positive, 35 ER just positive, and 43 both positive. In contrast, positive spasm was documented in 172 left coronary arteries (LCA) including 94 ACh just positive, 28 ER just positive, and 50 both positive. ACh provoked spasm more distally and diffusely, while ER induced spasm more proximally and totally or focally in the RCA. In the LCA, ACh provoked spasm more proximally, whereas ER induced spasm more distally. ER testing after the negative ACh tests of RCA and LCA documented new positive spasms in 10.3% (35/340) and 7.4% (28/376), respectively. Coronary artery trees may each have a sensitive receptor on each segment. We recommend the supplementary use of ACh and ER to document coronary artery spasm in the cardiac catheterization laboratory.