BACKGROUND: Little is known about the differences in left atrial (LA) volume and strain assessed with two-dimensional (2DE) and three-dimensional echocardiography(3DE). Therefore, this study aimed to compare 2DE and 3DE parameters in healthy subjects. METHODS: We obtained LA volumes and strains by 3DE and 2DE in 328 healthy subjects (52 ± 13 years; 51.83% men). RESULTS: Means of 3DE LA volume indexes (LAVIs) were higher than those derived from 2DE (maximal LAVI: 26.75 vs. 22.66 mL/m2; minimal LAVI: 11.76 vs. 9.30 mL/m2; preA LAVI: 19.52 vs. 16.23 mL/m2, respectively, all p < 0.001). Conversely, means of 3DE LA phasic strains were lower than those derived from 2DE (LA reservoir strain: 25.16 vs. 32.01%; LA conduit strain: 13.09 vs. 16.52%; LA contractile strain: 12.12 vs. 15.52%, respectively, all p < 0.001). Similarly, total, passive, and active LA emptying fractions (EF) were lower with 3DE than those with 2DE (all p < 0.05). Correlation between 3DE and 2DE was moderate for LAVIs (all r > 0.65, all p < 0.001), with 2DE showing a systematic underestimation (bias: -4.09 mL/m2 for maximal LAVI; -2.46 mL/m2 for minimal LAVI, -3.30 mL/m2 for preA LAVI). For LA strain parameters, only conduit strain showed moderate correlation (r = 0.65, p < 0.001) with an overestimation bias of +3.42%, while reservoir and contractile strains correlated poorly between modalities with wide limits of agreement. LAEFs also demonstrated poor inter-modality agreement. CONCLUSIONS: 2DE yielded lower LAVIs but higher LA strain and EF values compared to 3DE. Although there was a significant correlation between the 2D and 3D parameters, agreement analysis indicated that their normal reference values were not interchangeable.