We describe a method to construct tandem repeats of coding sequences for polypeptides interspersed by single methionine residues and terminating in a His(6) tag that can be purified by Ni chelate chromatography and then cleaved by cyanogen bromide (CNBr) into homogeneous peptide units, Annealing and unidirectional ligation of complementary 42mer oligonucleotides encoding the 13 amino acid residue yeast alpha-mating factor (alpha F) followed by ligation of the oligomerized 42mers into a pET vector placed the alpha-factor tandem repeats downstream of the ketosteroid isomerase (KSI) gene and upstream of a His(6) cassette. A KSI-(alpha F)(5)-His(6) fusion was overproduced in Escherichia coli, purified by Ni chelate chromatography, and then cleaved with CNBr to release insoluble KSI, the His(6) tail, and the alpha-factor peptide units, each terminating with homoserine (HS) lactone. HPLC yielded pure peptide in a yield of 56 mg/L. The alpha-factor-HS(lactone) could be ammonolyzed or hydrolyzed to yield alpha-factor-HS-amide or alpha-factor-HS, respectively. The alpha-factor-HS peptide had similar biological potency as authentic alpha-factor in yeast cell arrest assays. The alpha-factor-HS(lactone) was also reacted with a number of other compounds including analogs of fluorescein, dansyl, and biotin to produce alpha-factor peptides derivatized exclusively at the C-terminus. To test the ability of the expression system to produce longer peptides, 60-67 amino acid residue peptides encoding the Gla domain of profactor IX (FIXQS, FIXQS-His(6)) were also produced. Yields of 50-55 mg/L of pure FIXQS-His(6) and FIXQS-HS(lactone) were obtained.