The structural-functional organization of ammonia and glutamine metabolism in the liver acinus involves highly specialized hepatocyte subpopulations such as glutamine producing perivenous scavenger cells. However, it is still unclear whether this cell population is homogeneous and involves further subpopulations. This was investigated in the present study by proteome profiling of periportal glutamine synthetase-negative hepatocytes and perivenous glutamine synthetase (GS) expressing scavenger cells isolated from mouse and rat liver. Apart from established markers of GS+ hepatocytes such as glutamate transporter 1 (GLT1), ornithine aminotransferase (OAT) or ammonium transporter Rh type B (RHBG), we identified novel scavenger cell-specific proteins such as the basal transcription factor 3 (BTF3) and the heat-shock protein 25 (HSP25). Interestingly, BTF3 and HSP25 were heterogeneously distributed among GS+ hepatocytes as shown by immunofluorescence analyses in mouse, rat and human liver slices. Feeding experiments showed that RHBG but not GS protein levels were increased in the liver of mice fed with a high protein diet compared to standard chow. While the spatial distribution of GS and carbamoylphosphate synthetase-1 (CPS1) was not affected, periportal areas constituted by GLS2 positive hepatocytes were enlarged or reduced in response to high or low protein diet, respectively. The data suggest that the population of perivenous GS+ scavenger cells is heterogeneous and not uniform as previously suggested which may reflect a functional heterogeneity, possibly relevant for liver regeneration.