The production of
tRNA(
iMet) during Friend cell erythroid differentiation has been studied. In vitro measurements of total
nuclear RNA synthesis in nuclei isolated from Friend cells at different stages of differentiation show the total
RNA synthesis increases 1.5-fold at day 1 of induction and then decreases through days 2 and 3 to approximately 75% of its rate of synthesis in the nuclei of uninduced cells. The synthesis of
RNA polymerase III transcripts undergoes a similar fluctuation through day 2 of induction, but increases again at day 3. The specific synthesis of
tRNA(
iMet) was measured by hybridization of labelled
nuclear RNA to a
tRNA(
iMet) gene probe. During erythroid differentiation the percentage of
nuclear RNA represented by
tRNA(
iMet) remains constant (0.065%), so that the absolute synthesis of
tRNA(
iMet) fluctuates during differentiation, in the fluctuations in the synthesis of total
nuclear RNA. The relative synthesis of
tRNA(
iMet) in vivo was studied by labelling cells with 32Pi, isolating the resulting radioactive tRNA--5S
RNA population, and hybridizing this population to a
tRNA(
iMet) gene probe. The ratio of
tRNA(
iMet)/total tRNA--5S
RNA in newly synthesized cytoplasmic
RNA remains similar throughout differentiation (averaging 0.0171), implying that the fluctuations observed in the nuclear synthesis of
tRNA(
iMet) during differentiation probably also occur for the nuclear synthesis of most
tRNA and 5S
RNA species. Attempts were made to measure the relative steady-state concentration of
tRNA(
iMet) using both aminoacylation and in vitro end labelling of
tRNA followed by hybridization to a
tRNA(
iMet) gene probe. These two methods gave different results and we discuss the possible pitfalls of using enzymatic methods for quantitating
tRNA concentrations in the cell.