Procedures are described for the purification of the mitochondrial and cytosolic isozymes of phosphoenolpyruvate carboxykinase from rabbit liver. Examination of the purified isozymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated apparent homogeneity and identical molecular weights of approximately 65,000. Gel filtration chromatography of the native isozymes, however, yielded apparent molecular weights of 68,000 and 56,000 for the cytosolic and mitochondrial isozymes, respectively. The isoelectric points as determined by chromatofocusing were 5.8 for the mitochondrial isozyme and 5.0 for the cytosolic isozyme. The purified isozymes were readily separable on ion-exchange columns, with the cytosolic isozyme showing the greater affinity. A minor amount of cross-reactivity was apparent when each isozyme was immunotitrated with polyclonal antibodies raised in goat against the opposite isozyme. Peptide maps obtained by high pressure liquid chromatography of both tryptic digests and cyanogen bromide digests of the isozymes showed that many of the peaks were not coincident, suggesting that differences in the sequences are found throughout the primary structures of the isozymes.