Human Ab-secreting cell (ASC) populations in circulation are not well studied. In addition to B-1 (CD20+ CD27+ CD38 lo/int CD43+) cell and conventional plasmablast (PB)(CD20-CD27 hi CD38 hi) cell populations, in this study, we identified a novel B cell population termed 20+ 38 hi B cells (CD20+ CD27 hi CD38 hi) that spontaneously secretes Ab. At steady-state, 20+ 38 hi B cells are distinct from PBs on the basis of CD20 expression, amount of Ab production, frequency of mutation, and diversity of BCR repertoire. However, cytokine treatment of 20+ 38 hi B cells induces loss of CD20 and acquisition of CD138, suggesting that 20+ 38 hi B cells are precursors to PBs or pre-PBs. We then evaluated similarities and differences among CD20+ CD27+ CD38 lo/int CD43+ B-1 cells, CD20+ CD27 hi CD38 hi 20+ 38 hi B cells, CD20− CD27 hi CD38 hi PBs, and CD20+ CD27+ CD38 lo/int CD43− memory B cells. We found that B-1 cells differ from 20+ 38 hi B cells and PBs in a number of ways, including Ag expression, morphological appearance, transcriptional profiling, Ab skewing, Ab repertoire, and secretory response to stimulation. In terms of gene expression, B-1 cells align more closely with memory B cells than with 20+ 38 hi B cells or PBs, but differ in that memory B cells do not express Ab secretion-related genes. We found that B-1 cell Abs use Vh4-34, which is often associated with autoreactivity, 3-to 6-fold more often than other B cell populations. Along with selective production of IgM anti–phosphoryl choline, these data suggest that human B-1 cells might be preferentially selected for autoreactivity/natural specificity. In summary, our results indicate that human healthy adult peripheral blood at steady-state consists of three distinct ASC populations.