We have previously reported the discovery of new alkaloids such as pyrindicin, NA-337 A, etc., from Streptomyces sp. In the present communication, we report the isolation of TM-64, a new sulfur-containing basic substance that is positive to Dragendorff's reagent, from a thermophilic actinomycete, Thermoactinomyces strain No. TM-64. The strain was cultured at 45°C in appropriate media, and TM-64 was isolated from the culture filtrate via a series of solvent extractions (butyl acetate, HCl, CHCl3, ethyl ether) and recrystallized from benzene to obtain pure crystals. Physicochemical properties of TM-64 include a melting point of 120–122°C, elemental analysis (found: C 61.37%, H 5.89%, N 18.04%, S 9.70%; calculated for C16H18N4SO: C 61.12%, H 5.77%, N 17.82%, S 10.19%), a mass spectrum with an M+ ion at m/e 314.1183 (calculated for C16H16N4SO: 314.1190), a specific rotation of [α]₂₀ᴰ -6.0° (c 1, MeOH), UV maxima in methanol at 223 (ε 57150), 275 (10500), 282 (10500), and 291 (8350) nm, and an IR spectrum showing an amide absorption at 1630 cm⁻¹. TM-64 gave positive reactions to Dragendorff's, Ehrlich's, ninhydrin, and Mayer's reagents as well as a sulfur test, suggesting it has an indole skeleton and a sulfur-containing moiety. Pharmacological testing revealed a weak deteriorated corneal reflex in guinea pigs when a 3% TM-64 solution was applied to their eyes. The strain, which grows above 37°C (optimal at 45°C), forms white powdery aerial mycelium, bears single spores on substrate and aerial mycelia, and has a type III cell wall with meso-DAP (no arabinose or galactose), was identified as Thermoactinomyces and deposited at the Fermentation Research Institute, Chiba, Japan (FERM-P No. 3076). Its detailed structure is currently under investigation.