This study aimed at assessing the susceptibility of different tooth types (molar/premolar), surfaces (buccal/lingual) and enamel depths (100, 200, 400 and 600 µm) to initial erosion measured by surface microhardness loss (&#x0394;SMH) and calcium (Ca) release. Twenty molars and 20 premolars were divided into experimental and control groups, cut into lingual/buccal halves, and ground/polished, removing 100 µm of enamel. The initial surface microhardness (SMH&#8320;) was measured on all halves. The experimental group was subjected to 3 consecutive erosive challenges (30 ml/tooth of 1% citric acid, pH 3.6, 25°C, 1 min). After each challenge, &#x0394;SMH and Ca release were measured. The same teeth were consecutively ground to 200, 400 and 600 µm depths, and the experimental group underwent 3 erosive challenges at each depth. No difference was found in SMH&#8320; between experimental and control groups. Multivariate nonparametric ANOVA showed no significant differences between lingual and buccal surfaces in &#x0394;SMH (p = 0.801) or Ca release (p = 0.370). &#x0394;SMH was significantly greater in premolars than in molars (p < 0.05), but not different with respect to enamel depth. Ca release decreased significantly with increasing depth. Regression between Ca release and &#x0394;SMH at 100 µm depth showed lower slope and r² value, associated with greater Ca release values. At 200-600 µm depths, moderately large r² values were observed (0.651-0.830). In conclusion, different teeth and enamel depths have different susceptibility to erosion, so when Ca release is used to measure erosion, the depth of the test facet in enamel should be standardized, whereas this is less important if &#x0394;SMH is used.