In sediments, methane-oxidizing bacteria live in opposing gradients of methane and oxygen. In such a gradient system, the fluxes of methane and oxygen are controlled by diffusion and consumption rates, and the rate-limiting substrate is maintained at a minimum concentration at the layer of consumption. Opposing gradients of methane and oxygen were mimicked in a specific cultivation set-up in which growth of methanotrophic bacteria occurred as a sharp band at either c. 5 or 20 mm below the air-exposed end. Two new strains of methanotrophic bacteria were isolated with this system. One isolate, strain LC 1, belonged to the Methylomonas genus (type I methantroph) and contained soluble methane mono-oxygenase. Another isolate, strain LC 2, was related to the Methylobacter group (type I methantroph), as determined by 16S rRNA gene and pmoA sequence similarities. However, the partial pmoA sequence was only 86% related to cultured Methylobacter species. This strain accumulated significant amounts of formaldehyde in conventional cultivation with methane and oxygen, which may explain why it is preferentially enriched in a gradient cultivation system. © 2006 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.