An approach is presented for the detection of functional genes on chromosomal DNA in prokaryotes by two-pass tyramide signal amplification–fluorescence in situ hybridization (two-pass TSA-FISH). Functional genes are hybridized with 2,4-dinitrophenol (DNP)-labeled polynucleotide probes or digoxigenin-labeled oligonucleotide probes. Horseradish peroxidase (HRP)-labeled antibody is then immunologically bounded, and a first round of TSA with DNP-labeled tyramide is carried out. After the second immunological reaction with HRP-labeled anti-DNP antibody, cells hybridized with the probes are detected upon a second round of TSA with fluorescent-labeled tyramide. As a case study, we describe the use of two-pass TSA-FISH to detect the methanogenesis marker gene mcrA, which encodes the alpha subunit of methyl coenzyme M reductase in methanogenic archaea. Practical suggestions for using the two-pass TSA-FISH method are presented as well.