Nuclear RNA precursors to the mRNA for rat cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) and nuclear RNA species smaller in size than the mature enzyme mRNA have been identified by using hybridization probes specific for introns and exons from the gene. A number of processing intermediates, including an intact intron, were noted. Based on the size and distribution of the precursors observed, we propose a pathway for phosphoenolpyruvate carboxykinase RNA processing that involves splicing of introns starting at the 5' end of the RNA transcript and proceeding toward the 3' end.