In a previous study, we reported that Streptomyces tsukubaensis No. 9993 produces FK-506, a novel potent immunosuppressant. During FK-506 production, two minor components were isolated from the culture broth of S. tsukubaensis No. 9993 (deposit No. FERM BP-9272). Both components suppress in vitro immune responses and are structurally related to FK-506. One component, purified via preparative HPLC, was identified as FR-900520 produced by Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 through spectroscopic analysis. The other component was designated FR-900525, whose structure differs from FK-506 by replacing the piperidine-2-carboxylic acid moiety with proline. This paper describes the fermentation of S. tsukubaensis, and the isolation, characterization, and biological properties of FR-900525. FR-900525 has the molecular formula C43H67NO12, with a secondary ion (SI)-MS m/z of 790 (M+1), a melting point of 85–89°C, and a specific rotation [α]D23 of -88° (c 1.0, CHCl3). Acid hydrolysis confirmed proline (instead of FK-506's pipecolic acid) via TLC and amino acid analysis. Biological evaluations showed FR-900525 had an IC50 of 1.9 nM in a mouse mixed lymphocyte reaction, no cytotoxicity against EL-4 lymphoma cells at concentrations less than 3200 nM, an MIC of 0.5 μg/ml against Aspergillus fumigatus IFO 5840, and no adverse effects when administered intraperitoneally to ddY mice at 100 mg/kg.