Our understanding of the mammalian inwardly-rectifying (Kir) family of potassium channels has recently been advanced by X-ray crystal structures of two homologous prokaryotic orthologs (KirBac1.1 and KirBac3.1). However, the functional properties of these KirBac channels are still poorly understood. To address this problem we have cloned and characterised genes encoding KirBac orthologs from a wide variety of different prokaryots and a simple unicellular eukaryot. The functional properties of these KirBacs were then examined by growth complementation in a K⁺-uptake deficient strain of E.coli (TK2420). Whilst some KirBac genes exhibited robust growth complementation, others either did not complement, or showed temperature dependent complementation including KirBac1.1 and KirBac3.1. In some cases KirBac expression was also toxic to the growth of E.coli. The KirBac family exhibited a range of sensitivity to the K⁺ channel blockers Ba²⁺ and Cs⁺ as well as differences in their ability to grow on very low K⁺ media, thus demonstrating major differences in their permeation properties. These results reveal the existence of a functionally diverse superfamily of microbial KirBac genes and present an excellent resource for the structural and functional analysis of this class of potassium channels. Furthermore, the complementation assay used in this study provides a simple and robust method for the functional characterisation of a range of prokaryotic potassium channels which are difficult to study by traditional methods.