The mRNA coding for the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was partially purified from the liver of cyclic-AMP-treated rats by a procedure involving multiple oligo(dT)-cellulose chromatographies and sucrose gradient fractionations. The purification was monitored by translational assay using a wheat germ extract. Relative to RNA bound once to oligo(dT)-cellulose, the final material was enriched 20-fold in template activity for phosphoenolpyruvate carboxykinase synthesis. With this RNA preparation, cell-free enzyme synthesis amounted to 5% of total mRNA-directed protein synthesis. The apparent sedimentation coefficient of phosphoenolpyruvate carboxykinase mRNA in sucrose gradients was between 20 and 22 S, corresponding to an average molecular weight of 0.93 X 10(6). By formamide/polyacrylamide gel electrophoresis the molecular weight of the enzyme mRNA was estimated at between 0.91 X 10(6) and 1.12 X 10(6). From these estimates, it was concluded that considerable non-coding sequence(s) are present in the mRNA. Approximately 20% of the enzyme mRNA in rat liver failed to bind to oligo(dT)-cellulose, presumably because of the absence of a poly(A) segment. The translation of phosphoenolpyruvate carboxykinase mRNA by the wheat germ extract was inhibited in the presence of 7-methylguanosine 5'-phosphate. The enzyme mRNA appears therefore to have a 'cap' at the 5' end.