Unspecific peroxygenase (EC 1.11.2.1)represents a group of secreted heme-rnthiolate proteins that are capable of catalyzing the selective mono-oxygenation ofrndiverse organic compounds using only H2O2 as a cosubstrate. In this study, the peroxygenase from Agrocybe aegerita(AaeUPO)was found to catalyze the hydroxylation of various linear (e.g n-hexane), branched (e.g. 2,3-dimethylbutane) and cyclic alkanes (e.g. cyclohexane). The size of n-alkane substrates converted by AaeUPO ranged from gaseous propane (C3) to n-hexadecane (C16). Theyrnwere mono-hydroxylated mainly at the C2 and C3 position, rather than at the terminal carbon, and the corresponding ketones were formed as a result of overoxidation. In addition, a number of alkenes were epoxidized by AaeUPO,rnincluding linear terminal (e.g. 1-heptene), branched (2-methyl-2-butene) and cyclicrnalkenes (e.g. cyclopentene), as well as linear and cyclic dienes (buta-1,3-diene,cyclohexa-1,4-diene). Furthermore, the conversion of terminal alkynes (e.g. 1-octyne) gave the corresponding 1-alkyn-3-ol in low yield. Some of the reactions proceeded with complete regioselectivity and - in the case of linear alkanes, terminal linear alkenes and alkynes - with moderate to high stereoselectivity. Thernconversion of n-octane gave (R)-3-octanol with 99% enantiomeric excess (ee) and the preponderance of the (S)-enantiomer reached up to 72% ee of the epoxide product for the conversion of 1-heptene. Catalytic efficiencies (kcat/Km) determined for the hydroxylation and respectively epoxidationrnof the model compounds cyclohexane and 2-methyl-2-butene were 2.0 × 103 M-1 s-1 and 2.5 × 105 M−1 s−1. The results obtained in the deuterium isotope effect experiment with semi-rndeuterated n-hexane and the radical clock experiment with norcarane clearly demonstrated that the hydroxylation of alkanes proceeds via hydrogen abstraction, the formation of a substrate radical and a subsequent oxygen rebound mechanism. Moreover, stopped-flow experiments and substrate kinetics provedrnthe involvement of a porphyrin radical cation species (compound I; AaeUPO-I) as reactive intermediate in the catalytic cycle of AaeUPO, similar to other heme-thiolate enzymes (e.g. cytochrome P450 monooxygenases, P450s).