Biochemical and structural studies of plant phytochromes have been limited until recently by the difficulty of producing them in large amounts and as chromophore-bound, photochemically competent holoproteins in bacterial expression systems. This chapter provides protocols to achieve high-level production of the photosensory modules of plant phytochrome B in Escherichia coli. Protocols for codon optimization, designing multiple constructs, coexpression of the bilin chromophore synthesizing enzymes, and optimizing expression vectors, expression strains, and growth conditions are also included. These protocols can be easily adopted to overcome frequently encountered problems during heterologous production of eukaryotic phytochromes in Escherichia coli.