A microscale biosensor for continuous measurement of methane partial pressure based on a novel counterdiffusion principle is presented. Methane-oxidizing bacteria placed in the microsensor utilize oxygen from an internal oxygen reservoir when methane from the exterior diffuses through the tip membrane. The transducer is an internal oxygen microsensor with its tip positioned between the oxygen reservoir and the sensor tip membrane. The external partial pressure of methane determines the rate of bacterial oxygen consumption within the sensor, which in turn is reflected by the signal from the transducer. Tip diameters were down to 20 μm, enabling us to study methane distribution on a microscale. The microscale construction also results in a low stirring sensitivity and a 95% response time down to 20 s. By tailoring the geometry, sensors can be made to exhibit a linear response in the full range of 0-1 atm partial pressure of methane or, alternatively, to exhibit a linear response only at lower concentrations, improving the sensitivity to below 0.1 kPa, corresponding to ∼1 μM in aqueous solution. Temperature, oxygen, and H(2)S interfere with the signal; no interferences were detected from H(2), NH(3), CO(2), or acetate.