Background: Atherosclerotic plaques are heterogeneous with respect to inflammation, calcification, vascularity, oxygen, and temperature. We hypothesized that they also vary in pH and measured pH in living human carotid endarterectomized atherosclerotic plaques (CEA), Watanabe heritable hyperlipidemic (WHHL) rabbit aortas and human umbilical arteries (HUA). Methods and results: We measured pH of CEA of 48 patients, nine WHHL rabbit aortas and 11 HUA specimens (as controls) using a glass type microelectrode mounted on a micromanipulator in a 37°C incubator. We also used single emission and also dual emission fluorescence ratio imaging microscopy employing pH-sensitive probes to confirm pH heterogeneity. Mean pH measured at 415 points of CEA was 7.55±0.32; at 275 points of WHHL rabbit aortas it was 7.40±0.43; and in 233 points of HUA it was 7.24±0.1. In CEA, pH of yellow (lipid-rich) areas was significantly lower than pH in calcified areas (7.15±0.01 vs. 7.73±0.01, P<0.0001). The coefficients of variation (heterogeneity) of pH in CEA, WHHL rabbit aortas, and HUA were 0.038±0.010, 0.039±0.007, and 0.009±0.003, respectively (P=0.0001). Fluorescence microscopic imaging confirmed pH heterogeneity in both humans and rabbits but not in HUA. In a variance components analysis 82% of the heterogeneity was due to the within-plaque variation and 2% was attributable to between-plaque variation. Conclusions: Our findings support the hypothesis of pH heterogeneity in plaques, and suggest a possible role for detecting low pH in the detection of plaque vulnerability. The source of pH heterogeneity particularly acidic pH, its impact on the stability of plaques and its potential clinical utility in locating vulnerable plaques remain to be evaluated. © 2002 Elsevier Science Ireland Ltd. All rights reserved.