A novel valanimycin-resistance determinant (vImF) was isolated from a cosmid containing Streptomyces viridifaciens DNA that leads to valanimycin production in Streptomyces lividans. Expression of the vImF gene in both Escherichia coli and S. lividans provided valanimycin resistance. The nucleotide sequence of vImF consists of 1206 bp and the deduced amino acid sequence encodes a polypeptide with 12 putative transmembrane-spanning segments and a calculated pI of 10.1. VImF shows significant similarities to other known or putative transmembrane efflux proteins that confer antibiotic resistance, but it appears to be specific for valanimycin. The sequence similarities suggest that VImF is a member of the DHA12 family within the major facilitator superfamily of transport proteins and that it is probably involved in active valanimycin efflux energized by a proton-dependent electrochemical gradient.