One of the current limitations in genetic manipulations of streptomycetes and other actinobacteria is the lack of negative selection markers that can be used without the introduction of specific mutations in the host strain. We here report the development and application of a counterselection system based on cytosine deaminase (CodA). This enzyme can transform 5-flurocytosine (5-FC) into 5-fluorouracil (5-FU), a compound highly toxic for most living cells. Among sixteen actinobacterial species tested, all were resistant to 5-FC, most being sensitive to 5-FU suggesting that they are naturally devoid of cytosine deaminase. We propose a series of novel vectors for actinobacteria that include a synthetic gene encoding the CodA protein from Escherichia coli optimized for expression in Streptomyces. When CodA-positive cells are exposed to 5-FC, they are subject to a potent negative selection pressure. Gene disruptions and the introduction of an unmarked in-frame deletion were successfully achieved with these vectors. The D314A-mutated version of this gene (codASM) is particularly useful as it requires only low levels of 5-FC (20 - 50 micrograms per ml) for counterselection.