Two distinguishable activity bands for dye‐linked l‐proline dehydrogenase (PDH1 and PDH2) were detected when crude extract of the hyperthermophilic archaeon Pyrococcus horikoshii OT‐3 was run on a polyacrylamide gel. After purification, PDH1 was found to be composed of two different subunits with molecular masses of 56 and 43 kDa, whereas PDH2 was composed of four different subunits with molecular masses of 52, 46, 20 and 8 kDa. The native molecular masses of PDH1 and PDH2 were 440 and 101 kDa, respectively, indicating that PDH1 has an α4β4 structure, while PDH2 has an αβγδ structure. PDH2 was found to be similar to the dye‐linked l‐proline dehydrogenase complex from Thermococcus profundus, but PDH1 is a different type of enzyme. After production of the enzyme in Escherichia coli, high‐performance liquid chromatography showed the PDH1 complex to contain the flavins FMN and FAD as well as ATP. Gene expression and biochemical analyses of each subunit revealed that the β subunit bound FAD and exhibited proline dehydrogenase activity, while the α subunit bound ATP, but unlike the corresponding subunit in the T. profundus enzyme, it exhibited neither proline dehydrogenase nor NADH dehydrogenase activity. FMN was not bound to either subunit, suggesting it is situated at the interface between the α and β subunits. A comparison of the amino‐acid sequences showed that the ADP‐binding motif in the α subunit of PDH1 clearly differs from that in the α subunit of PDH2. It thus appears that a second novel dye‐linked l‐proline dehydrogenase complex is produced in P. horikoshii.