Abstract Description Although thought to be a distinct homogeneous subset, B-1a cells are heterogeneous. We investigated the transcriptional and clonal heterogeneity of peritoneal cavity B cells by performing single-cell RNA sequencing and BCR repertoire profiling. Initial clustering confirmed three major populations: B1a, B1b, and B2 cells. As expected, the B1a cell population showed highly repetitive BCRs whereas most BCR clonotypes of B1b and B2 cells were found only once. Interestingly, we could observe two distinct B1a subpopulations biased towards IgH V11 or V12, both of which are associated with autoreactive specificities binding to phosphatidylcholine. The two B1a cell subpopulations were also different based on the gene expression profiles as the two subpopulations persisted when we regrouped them after removing the contribution of immunoglobulin heavy chains. We could also identify the two subpopulations by flow cytometry with identified cell surface markers including CD11b, B220, and ICAM-1. The two subpopulations largely maintained their properties upon adoptive transfer experiments, but some of Fucosyl transferase 8(-) B1a cells were converted into Fucosyl transferase 8(+) B1a cells whereas the reverse conversion was not observed. In summary, we identified two distinct B1a cell subpopulations based on the usage of BCR repertoire and gene expression profiles. Their functional differences may help define human B-1a cells and their differential contribution to humoral immunity. Funding Sources Supported by RS-2024-00405650, 2023R1A2C2004510 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)