A recombinant bacterial expression system that generates (13)C-labeled heme or 15 N-labeled heme in functional cytochrome P450 enzymes and other heme-containing systems is reported here using a mutant strain of Escherichia coli (HU227) in which the HemA gene is inactive. By synthesizing several isotopomers of aminolevulinic acid with (13)C or (15)N at different locations, isotopes have been incorporated with high abundance into the heme cofactor of five different cytochrome P450 isoforms, along with one peroxidase. Confirmed both (13)C- and (15)N-incorporation; spectral and catalytic assays show the labeled enzymes produced in this system are functional.