The yeast a-factor mating peptide and its transporter Ste6 are normally expressed only in MATa haploid cells. The a-factor is initially produced as a 36- or 38-residue peptide precursor and must undergo extensive post-translational processing to produce an active 12 amino-acid lipopeptide. To better understand the steps required for Ste6-dependent a-factor transport, we have reconstituted a-factor synthesis and transport in MATalpha haploids and MATa/alpha diploids. Ste6 and a-factor were stably expressed in MATalpha and MATa/alpha cells and the ectopically expressed a-factor was correctly processed. In addition, Ste6 was able to transport a-factor from all cell types, indicating that once expressed no other MATa-specific functions are required. However, despite significant levels of a-factor secretion, MATalpha cells are unable to support efficient mating.