The dose-response characteristics of initiation of hepatocarcinogenesis by diethylnitrosamine (DEN) was investigated in the neonatal female rat by means of the quantitative stereologic estimation of altered hepatic foci (AHF) expressing multiple markers. At 5 days of age, female Sprague-Dawley rats were given a single i.p. dose of DEN (0.1-30 mg/kg body wt) or the vehicle (trioctanoin). The semisynthetic AIN-76A diet was provided to half of the rats in each treatment group, while the remainder received this diet containing 500 mg phenobarbital (PB)/kg for 8 months from weaning until the animals were killed. To ascertain more exactly the dose-response relationship for initiation by DEN, the number, volume percentage and phenotypes of the resulting AHF were determined by quantitative stereological analysis on serial sections of frozen tissue, each stained for one of four markers of preneoplasia. A linear relationship was observed between the dose of DEN (0-30 mg/kg) and the number and volume percentage of AHF detected, with each single marker or the total number of AHF detected when the placental isozyme of glutathione S transferase, gamma-glutamyl transpeptidase (GGT), canalicular adenosine triphosphatase, or glucose-6-phosphatase was used as the marker. For each dose, PB administration increased the number and volume of AHF scored compared with similarly initiated rats that did not receive a promoting stimulus. This was, in part, owing to enhanced GGT expression in AHF with PB administration. Promotion by PB resulted in a distribution of AHF phenotypes altered from that observed in rats not receiving PB. Initiation of AHF in neonatal female rats by DEN was linear with doses from 0 to 30 mg/kg for all four of the phenotypic markers employed. In addition, while PB administration stimulated the growth of all AHF phenotypes, the growth of a subset of AHF that expressed the widest variation in preneoplastic markers was specifically enhanced by PB administration.