In the course of screening for new antibiotics active against P388 murine leukemia cells, novel antibiotics pyrisulfoxin A (1) and pyrisulfoxin B (2) were isolated from the culture broth of Streptomyces californicus BS-75. The producing organism was cultivated, and the antibiotics were isolated via processes including centrifugation, column chromatography (Dowex 1, Diaion HP-20, ODS column) and preparative HPLC, yielding colorless powders of 1 (6.5mg) and 2 (1.6mg). The molecular formula of 1 was determined as C13H13N3O3S by HRFAB-MS. Its structure was elucidated by comparing 13C and 1H NMR spectral data with caerulomycin A, revealing a methylsulfoxide group at the C-5 position and an anti configuration of the oxime group. The molecular formula of 2 was C13H11N3O2S; its IR spectrum showed a cyano group characteristic absorption, and NMR data indicated absence of oxime protons. Pyrisulfoxin A exhibited cytotoxicity against P388 murine leukemia cells with an IC50 value of 0.1 μg/ml, while pyrisulfoxin B showed no cytotoxicity. The cytotoxic activity of 1 was comparable to that of collismycin A and SF2738A.