The stability and response of histochemical phenotypes of altered hepatic foci (AHF) were studied both in the presence and following the withdrawal of 0.05% phenobarbital (PB) treatment in rats previously given a single dose of diethylnitrosamine (DEN) 20-24 h following partial hepatectomy (PH). AHF were scored by their expression of three biochemical markers: gamma-glutamyl transpeptidase (GGT), adenosine triphosphatase and glucose-6-phosphatase (G6P). AHF demonstrated significant heterogeneity with respect to the marker alterations. The use of three markers in the present study confirmed the findings of our earlier study, which showed the maximal response of GGT+ AHF to PB administration following PH/DEN initiation and the stability of GGT+/AHF induced by the PH/DEN/PB regimen after the withdrawal of PB. In the regimen employed, the GGT marker alone scored the great majority of the AHF detected by all three markers. The frequency distribution of histochemical phenotypes remained relatively constant in AHF during continuous PB administration and in AHF promoted by PB followed by a 6-month period of feeding a diet containing no PB. These findings suggest that individual AHF remain phenotypically stable throughout the PB promotion phase, i.e., do not progress from one phenotype to another. In every marker class, the mean volume of AHF increased during continuous PB administration. These data illustrate the enhancing effect of PB on the growth of the AHF. The size of AHF continued to increase following the withdrawal of PB in the 3-month PB treatment group, but not in the animals treated for 4 months. A mechanism that may account for the differences in these two treatment groups is discussed.