High aldehyde dehydrogenase (ALDH ^hi ) activity has been reported in normal and cancer stem cells. We and others have shown previously that human ALDH ^hi cardiac atrial appendage cells are enriched with stem/progenitor cells. The role of ALDH in these cells is poorly understood but it may come down to the specific ALDH isoform(s) expressed. This study aimed to compare ALDH ^hi and ALDH ^lo atrial cells and to identify the isoform(s) that contribute to ALDH activity, and their functional role. <b>Methods and Results:</b> Cells were isolated from atrial appendage specimens from patients with ischemic and/or valvular heart disease undergoing heart surgery. ALDH ^hi activity assessed with the Aldefluor reagent coincided with primitive surface marker expression (CD34 ⁺ ). Depending on their ALDH activity, RT-PCR analysis of ALDH ^hi and ALDH ^lo cells demonstrated a differential pattern of pluripotency genes (Oct 4, Nanog) and genes for more established cardiac lineages (Nkx2.5, Tbx5, Mef2c, GATA4). ALDH ^hi cells, but not ALDH ^lo cells, formed clones and were culture-expanded. When cultured under cardiac differentiation conditions, ALDH ^hi cells gave rise to a higher number of cardiomyocytes compared with ALDH ^lo cells. Among 19 ALDH isoforms known in human, ALDH1A3 was most highly expressed in ALDH ^hi atrial cells. Knocking down ALDH1A3, but not ALDH1A1, ALDH1A2, ALDH2, ALDH4A1, or ALDH8A1 using siRNA decreased ALDH activity and cell proliferation in ALDH ^hi cells. Conversely, overexpressing ALDH1A3 with a retroviral vector increased proliferation in ALDH ^lo cells. <b>Conclusions:</b> ALDH1A3 is the key isoform responsible for ALDH activity in ALDH ^hi atrial appendage cells, which have a propensity to differentiate into cardiomyocytes. ALDH1A3 affects <i>in vitro</i> proliferation of these cells.