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Background Fluorescent proteins are used widely as reporter genes in many organisms. We previously codon-optimized mCherry for Mycobacterium tuberculosis and generated expression constructs with high level expression in mycobacteria with multiple uses in vitro and in vivo. However, little is known about the expression of fluorescent proteins in mycobacteria and the translational start codon for mCherry has not been experimentally determined. Results We determined the translational start site for functional (fluorescent) mCherry in mycobacteria. Several potential translational start codons were identified; introduction of downstream stop codons by mutagenesis was used to determine which start codon was utilized in the bacterial cells. Fluorescent protein was expressed from a construct which would allow translation of a protein of 226 amino acids or a protein of 235 amino acids. No fluorescence was seen when a construct which could give rise to a protein of 219 amino acids was used. Similar results were obtained in mycobacteria and in Escherichia coli. Western blotting confirmed that mCherry was expressed from the constructs encoding 235 or 226 amino acids, but not from the plasmid encoding 219 amino acids. N-terminal sequencing and mass determination confirmed that the mature protein was 226 amino acids and commenced with the amino acid sequence AIIKE. Conclusion We conclude that mCherry is expressed in M. tuberculosis as a smaller protein than expected lacking the GFP-derived N-terminal sequence designed to allow efficient fusions.