We have analyzed two mutations in src responsible for phenotypic reversion and subsequent retransformation of a Rous sarcoma virus-transformed rat cell. Comparison of the nucleotide sequences of cloned proviral DNAs reveals a single base pair insertion approximately 438 bp from the 5' end of src in the revertant line 000. This frameshift mutation accounts for an 18 kd protein as the prematurely terminated product of src. The mutation is suppressed in the retransformed line (000*) by a 242 bp duplication that corrects the reading frame to permit synthesis of a 68 kd src protein. A 43 kd src protein with a normal carboxyl terminus is also present in 000 and 000* cells. To make this protein, translation must begin at an internal AUG, found just upstream from the frameshift mutation; in both lines, two src proteins appear to be initiated independently from the same mRNA species. Our results imply that a protein of 7 kd is synthesized from a second reading frame within wild-type src.