Maize mitochondria of cytoplasmic male sterile (cms-S) plants contain two linear episomes, S1 (6397 bp) and $2 (5453 bp). $1 contains three long open reading frames URF2 (1017bp), URF3 (2782bp) and URF4 (768 bp). We have demonstrated that the URF3 sequence of $1 encodes a protein with an apparent molecular weight of 103 kDa which is found in cms-S but undetectable in cms-T, cms-C or normal (fertile) mitochondria. A translational fusion containing the 5' terminus of the lacZ gene and 800 bp of the 3' end of URF3 was isolated from a cms-S mitochondrial genomic library in the expression vector 2gtl 1. Polyclonal antibodies raised against the resulting fusion protein immunoprecipitated a 103 kDa polypeptide from among [35S]-methionine-labeled cms-S mitochondrial proteins but not from normal mitochondrial proteins. The mitochondria of fertile F1 plants resulting from a cross between B37 cms-S and Ky21 (universal restorer) contain as much of this 103 kDa protein as is observed in sterile cms-S mitochondria. The mitochondria of fertile cytoplasmic revertants from cms-RD and cms-LM in a WF9 nuclear background also synthesized the 103 kDa protein. We conclude that the URF3 sequence of the SI episome is expressed in vivo and that the presence of its gene product in maize mitochondria is not sufficient to confer the male sterile phenotype.