We found ASR to be an effective method to generate eleven, self-sufficient, ancestral CYP116B P450 enzymes. A range of stabilities was observed, with a trend towards increasing thermostability along the branches leading to the more thermostable extant forms, suggesting that thermophilicity arose recently in the P450-TT and other extant thermophilic lineages, rather than being maintained from a highly stable ancestor. The inferred CYP116B ancestors were all catalytically active and showed unique regioselectivity profiles towards the hydroxylation of decanoic acid, including ancestors with novel selectivity for the ω-2 position. Finally, by examining the existing CYP116B structural information and comparing the ancestral sequences, we were able to identify two active site residues that were partially responsible for changes to the regioselectivity of the CYP116B ancestors vs. the wild-type P450-TT from terminal to mid-chain hydroxylation.