The present study aimed at investigating the in-vitro oxidation of acrylonitrile (ACN) to cyanide (CN-) by prostaglandin H synthase (PHS). Detection of CN- is considered a marker for free radical intermediates involved in ACN-induced toxicity. First, most favorable circumstances for ACN oxidation were characterized: pH (4.5), temperature (37ºC) and time of incubation (60 min.). In addition, the concentrations of ACN, PHS and H2O2 in incubation mixtures were assessed for further reaction characterization. The reaction maximum velocity (Vmax) was calculated to be 582.75 pmol CN-/mL/min and the Michaelis-Menten constant (Km) was 149.25 μmol ACN. Adding PHS inhibitors; resveratrol, quercetin, indomethacin or troloc-C to the reaction mixtures significantly reduced the rate of ACN oxidation. In conclusion, the present study demonstrates the ability of PHS to oxidize ACN to CN- and provides a clue for the explanation of ACN target toxicity.