Liver sinusoids contain a population of large granular lymphocytes or natural killer cells, originally termed pit cells. After isolation and purification, these cells were separated into a low-density and a high-density fraction. The liver low-density fraction differs significantly in morphology and function from cells of the blood, whereas the liver high-density fraction shows intermediate properties. In this study we demonstrate that this morphological and functional heterogeneity is based on subsequent steps of differentiation of the large granular lymphocytes within the liver. When cell proliferation was suppressed by sublethal total body irradiation, the life span of the hepatic large granular lymphocytes could be determined: high-density and low-density populations were totally depleted within 1 and 2 wk after irradiation, respectively. By using intravenous asialo-GM1 antiserum to deplete animals of asialo-GM1-positive cells, we found that the depletion of the asialo-GM1-positive cells preceded the depletion of asialo-GM1-negative hepatic low-density large granular lymphocytes by approximately 1 wk. Direct evidence that the asialo-GM1-positive high-density large granular lymphocytes are precursors of the low-density large granular lymphocytes was given by adoptive transfer experiments with fluorescent-labeled high-density cells. Three days after their injection, labeled large granular lymphocytes were found in the hepatic low-density fraction of the recipient rat, and these cells had developed morphological characteristics of low-density large granular lymphocytes. It is concluded therefore that marginating blood large granular lymphocytes differentiate through high-density large granular lymphocytes into the typical liver-specific low-density large granular lymphocytes or pit cells. (HEPATOLOGY 1993;18:919-925).