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 (FA1(T) and FA2(T)) formed a cluster together with Friedmanniella capsulata and Friedmanniella lacustris and the other two strains (FB1(T) and FB2(T)) formed a cluster together with Friedmanniella antarctica and Friedmanniella spumicola. The cell-wall peptidoglycan contained ll-A(2)pm 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-C(15 : 0)) and 13-methyltetradecanoic acid (i-C(15 : 0)). The major polar lipids were phosphatidylinositol and phosphatidylglycerol. In addition, strain FA1(T), FB1(T), and FB2(T) contained diphosphatidylglycerol and phosphatidylcholine. The DNA G+C contents were: 72 mol%, 73 mol%, 74 mol% and 75 mol% for strains FA1(T), FA2(T), FB1(T), and FB2(T), 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 FA1(T), FA2(T), FB1(T) and FB2(T) represent novel species of the genus Friedmanniella. Therefore, the names Friedmanniella luteola (type strain FA1(T)=DSM 21741(T)=NBRC 104963(T)), Friedmanniella lucida (type strain FA2(T)=DSM 21742(T)=NBRC 104964(T)), Friedmanniella okinawensis (type strain FB1(T)=DSM 21744(T)=NBRC 104966(T)) and Friedmanniella sagamiharensis (type strain FB2(T)=DSM 21743(T)=NBRC 104965(T)) are proposed for these new strains.