A hybrid plasmid, pJC74, carrying the large (3.35 Md) EcoRI-PstI fragment of ColE1, the 1.3 and 1.8 Md PstI fragments of plasmid R1 drd19 (containing a part of Tn3 and known to specify transpostion functions), and a part of gamma bacteriophage DNA carrying the fused cohesive ends of gamma (cos) was constructed. Supercoiled DNA of pJC74 and a series of deletions (series pJC75) were used in a cell-free coupled transcription-translation system. Analysis of the proteins produced allowed the identification of a 28 to 30 kd protein and/or a 3 kd protein responsible for mobilisation of the plasmids in sex-factor-promoted conjugation. Furthermore, three protein bands of 12, 12.5 and 13 kd were correlated with the presence of a portion of the Tn3 transposon previously shown to code for transposition functions which can complement in trans. These latter and the 30 kd beta-lactamase were the only proteins identified as Tn3-specific by comparison of ColE1 with RSF2124.