Aged female F344/NCr rats were exposed to phenobarbital (PB), 500 p.p.m. in the drinking water, from 26 months of age, for periods of 4, 8 or 9-27 weeks. In groups of control and PB-exposed rats sacrificed at these time periods the number and volume of basophilic and eosinophilic focal hepatocellular proliferative lesions (FHPL), including altered foci and adenomas in hematoxylin- and eosin-stained sections and FHPL identified histochemically by their content of gamma-glutamyl transpeptidase (GGT), were determined using computerized image analysis. Tritiated thymidine [3H]TdR was injected prior to sacrifice to determine labeling indices (LI) of normal hepatocytes and hepatocytes in FHPL. Rats receiving PB had significantly increased numbers and volumes of eosinophilic and GGT-positive FHPL while the numbers and volumes of the common basophilic FHPL seen in controls were not affected by PB exposure. The LI of all FHPL were higher than that of normal hepatocytes, but PB exposure did not affect the LI of basophilic FHPL. An uncommon GGT-positive FHPL found in control rats was detected in zone 1 of the hepatic acinus, a similar location of the GGT-positive eosinophilic FHPL seen in PB-exposed rats, while the common basophilic FHPL was observed in zones 1-3. These findings suggest that PB does not promote the growth or development of the common naturally occurring basophilic FHPL but either promotes the uncommon GGT-positive FHPL or induces new FHPL de novo.