To develop an in vitro phytochrome assembly system, we have expressed an oat phytochrome cDNA in both the yeast Saccharomyces cerevisiae and the bacterium Escherichia coli. Analysis of soluble protein extracts showed that the recombinant apophytochromes were full-length and capable of covalently attaching the phytochrome chromophore analogue phycocyanobilin. Difference spectra indicated that in vitro-assembled holophytochrome species were photoreversible; however, maxima and minima difference absorption values were blue-shifted relative to those of the native photoreceptor. Extracts containing the recombinant apophytochromes were also incubated with phytochromobilin, the natural chromophore synthesized from biliverdin by cucumber etioplast preparations. In these experiments, the difference spectrum obtained was identical to that of native oat holophytochrome. These results suggest that the recombinant apophytochromes adopt a structure similar to that of the apoprotein biosynthesized in vivo. ELISAs were used to quantitate phytochrome expression levels in both yeast and E. coli extracts. These measurements show that 62-75% of the phytochrome apoprotein in the soluble protein extract was competent to assemble with bilins to form spectrally active holophytochrome.