Controversy has arisen about the nature of circulating human CD20(+)CD27(+)CD43(+)CD70(-)CD69(-) B cells. Although originally described as being the human counterpart of murine B-1 B cells, some studies have raised the possibility that these might instead be plasmablasts. In this article, we have further characterized the putative B-1 cells and compared them directly with memory B cells and plasmablasts for several functional characteristics. Spontaneous antibody production of different isotypes as well as the induced production of antigen-specific antibodies after vaccination with a T-cell-dependent antigen did not reveal differences between the putative B-1 cells and genuine CD20(-) plasmablasts. Gene expression profiling of different B-cell subsets positioned the phenotype of putative B-1 cells closer to CD20(-) plasmablasts than to memory B cells. Moreover, putative B-1 cells could be differentiated into CD20(-) plasmablasts and plasma cells in vitro, supporting a pre-plasmablast phenotype. In conclusion, characterization of the putative B-1 cells revealed a functional phenotype and a gene expression profile that corresponds to cells that differentiate into CD20(-) plasmablasts. Our data offer perspectives for the investigation of differentiation of B cells into antibody secreting cells.