Four Gram-positive, non-motile, aerobic actinobacteria were isolated from spiders and their webs. Their genetic, phenotypic and chemical properties were studied. The 16S rRNA gene sequence data suggested that the four novel isolates belonged to the genus Friedmanniella. Two strains (FA1T and FA2T) formed a cluster together with Friedmanniella capsulata and Friedmanniella lacustris and the other two strains (FB1T and FB2T) formed a cluster together with Friedmanniella antarctica and Friedmanniella spumicola. The cell-wall peptidoglycan contained ll-A2pm and mycolic acids were absent. Isoprenoid quinones were mainly composed of MK-9(H4), MK-9(H2) and MK-9 and the predominant fatty acids were 12-methyltetradecanoic acid (ai-C15 : 0) and 13-methyltetradecanoic acid (i-C15 : 0). The major polar lipids were phosphatidylinositol and phosphatidylglycerol. In addition, strain FA1T, FB1T, and FB2T contained diphosphatidylglycerol and phosphatidylcholine. The DNA G+C contents were: 72 mol%, 73 mol%, 74 mol% and 75 mol% for strains FA1T, FA2T, FB1T, and FB2T, respectively. DNA–DNA hybridization studies demonstrated that the novel strains showed low relatedness values to F. capsulata, F. lacustris, F. antarctica and F. spumicola. These data support the proposal that strains FA1T, FA2T, FB1T and FB2T represent novel species of the genus Friedmanniella. Therefore, the names Friedmanniella luteola (type strain FA1T=DSM 21741T=NBRC 104963T), Friedmanniella lucida (type strain FA2T=DSM 21742T=NBRC 104964T), Friedmanniella okinawensis (type strain FB1T=DSM 21744T=NBRC 104966T) and Friedmanniella sagamiharensis (type strain FB2T=DSM 21743T=NBRC 104965T) are proposed for these new strains.