The relative rates of synthesis of two major tRNASer species in rooster liver were simultaneously assessed during induction by estradiol-17beta of the synthesis of a serine-rich phosphoprotein, vitellogenin. The relative rates of tRNA synthesis were determined by a double-label method in which nonspecific effects of the hormone were avoided. Isotope ratios of highly purified tRNASer species were measured following an in vivo labeling procedure which included a 7-day labeling period with [5-3H]orotic acid prior to, and a 6 h labeling with [6-14C]orotic acid from 42 h after the hormone injection. tRNASer (AGU,C) and tRNASer (UCU,C,A) were extensively purified by chromatography on benzoylated DEAE-cellulose in the presence and absence of Mg2+. In three separate labeling experiments the rate of tRNASer (UCU,C,A) synthesis was slightly but not significantly increased relative to the rate of tRNASer (AGU,C) synthesis during the period when vitellogenin was synthesized at a constant rate and the level of tRNASer continued to rise. The results suggest that mechanisms other than a differential rate of transcription are involved in the regulation of tRNASer levels in avian liver during vitellogenin induction.