Biodegradation of poly(cis-1,4-isoprene) (rubber) by Gram-negative bacteria has been investigated on the enzymatic level only in Steroidobacter cummioxidans 35Y (previously Xanthomonas sp. 35Y). This species produces two kinds of rubber oxygenases, RoxA(35Y) and RoxB(35Y), one of which (RoxB(35Y)) cleaves polyisoprene to a mixture of C-20- and higher oligoisoprenoids while the other (RoxA(35Y)) cleaves polyisoprene and RoxB(35Y)-derived oligoisoprenoids to the C-15-oligoisoprenoid 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). ODTD can be taken up by S. cummioxidans and used as a carbon source. Gram-positive rubber-degrading bacteria employ another type of rubber oxygenase, latex clearing protein (Lcp), for the initial oxidative attack of the polyisoprene molecule. In this contribution, we examined which type of rubber oxygenase is present in the only other well-documented Gram-negative rubber-degrading species, Rhizobacter gummiphilus NS21. No homologue for an Lcp protein but homologues for a putative RoxA and a RoxB protein (the latter identical to a previously postulated LatA-denominated rubber cleaving enzyme) were identified in the genome of strain NS21. The roxA(NS21) and roxB(NS21) genes were separately expressed in a roxA(35Y)/roxB(35Y) background of S. cummioxidans 35Y and restored the ability of the mutant to produce oligoisoprenoids. The RoxA(NS21) and RoxB(NS21) proteins were each purified and biochemically characterised. The resultsin combination with in silico analysis of databasesindicate that Gram-negative rubber-degrading bacteria generally utilise two synergistically acting rubber oxygenases (RoxA/RoxB) for efficient cleavage of polyisoprene to ODTD.