The aims of this study were to assess the effect of prone and supine positioning on 99Tc(m)-MIBI myocardial SPET images and the contribution of cardiac axis change. We compared 227 tomograms of patients imaged in the prone position with 227 tomograms of the same patients imaged in the supine position. For each tomographic session, the axis angle of the heart was recorded using an in-house program. The results showed a significant change in the cardiac axis angle of 9 degrees in the transaxial plane (P < 0.001). This change in the cardiac axis correlated with differences in cardiac wall activity (wall activity when the patient was imaged in the prone position minus wall activity when the patient was imaged in the supine position). Our results suggest that factors other than diaphragmatic movement and attenuation could account for the differences in wall activity observed when patients are imaged prone versus supine. Differences in the intensity of photon attenuation in the heart itself, depending on the cardiac axis, could be a contributing factor. Quantitation of the variation in wall activity leads us to suggest that 99Tc(m)-MIBI SPET should be performed in the prone position to allow better visualization of the inferior and the septal walls. The anterior and lateral walls are better studied in the supine position. Images acquired in both the prone and supine positions would allow the best assessment of all walls.