Rat NK cells are divided into major subsets expressing either Ly49 receptors or the inhibitory NKR-P1B receptor in conjunction with NKG2A/C/E receptors. A minor subset of NKp46 cells lacking expression of both Ly49 receptors and NKR-P1B is present in blood and spleen and is associated with decreased functional competence. We hypothesized that this subset may represent precursors to Ly49 and/or NKR-P1B NK cells. When cultured in vitro in IL-2 and IL-15 or adoptively transferred to syngeneic hosts, a portion of NKR-P1BLy49s3 cells transformed to express NKR-P1B, but very little Ly49s3. Acquisition of NKR-P1B by NKR-P1BLy49s3 cells coincided with increased degranulation. In addition, although NKR-P1BLy49s3 cells highly proliferate, proliferative activity was reduced upon acquisition of NKR-P1B at comparable levels to bona fide NKR-P1B NK cells. A fraction of NKR-P1BLy49s3 cells remained negative for NKR-P1B, both in vitro and after adoptive transfer in vivo. Most NKR-P1BLy49s3 cells expressed the transcription factor Eomesodermin and NK cell markers, indicating that these cells represent conventional NK cells. Our findings suggest that the NKR-P1BLy49s3 NK cells are precursors to NKR-P1B single-positive cells and that functional competence is acquired upon expression of NKR-P1B.