Bacillus megaterium flavocytochrome P450 BM3 (BM3) is a high activity fatty acid hydroxylase, formed by the fusion of soluble cytochrome P450 and cytochrome P450 reductase modules. Short chain (C6, C8) alkynes were shown to be substrates for BM3, with productive outcomes (i.e. alkyne hydroxylation) dependent on position of the carbon-carbon triple bond in the molecule. Wild-type P450 BM3 catalyses ω-3 hydroxylation of both 1-hexyne and 1-octyne, but is suicidally inactivated in NADPH-dependent turnover with non-terminal alkynes. A F87G mutant of P450 BM3 also undergoes turnover-dependent heme destruction with the terminal alkynes, pointing to a key role for Phe87 in controlling regioselectivity of alkyne oxidation. The terminal alkynes access the BM3 heme active site led by the acetylene functional group, since hydroxylated products are not observed near the opposite end of the molecules. For both 1-hexyne and 1-octyne, the predominant enantiomeric product formed (up to ∼90%) is the (S)-(-)-1-alkyn-3-ol form. Wild-type P450 BM3 is shown to be an effective oxidase catalyst of terminal alkynes, with strict regioselectivity of oxidation and potential biotechnological applications. The absence of measurable octanoic or hexanoic acid products from oxidation of the relevant 1-alkynes is also consistent with previous studies suggesting that removal of the phenyl group in the F87G mutant does not lead to significant levels of ω-oxidation of alkyl chain substrates.