The soluble cell-free fraction (150,000 g high-speed supernatants [HSS]) of Pseudomonas fluorescens NUMB 11764 contains putative cyanide oxygenase (CNO) responsible for initiating cyanide oxidation and assimilation as a nitrogenous growth substrate. CNO activity, assayed either by cyanide-dependent O-2 or NADH uptake, or by conversion of radioactive K-14 CN to (CO2)-C-14 was detected at micromolar concentrations (apparent half-saturation constant, 4 muM). Results demonstrating that CNO requires a protein-enriched cell fraction and a low MW redox factor (< 500 Da) for which reduced biopterin could substitute are presented. The properties of CNO are consistent with those of a pterin hydroxylase.