The proteinous antibiotic neocarzinostatin was succinylated using succinic anhydride, and two bis-succinyl compounds, neocarzinostatin derivatives (SUC-I and SUC-II), were obtained. The biological and chemical properties of SUC-I and SUC-II were investigated after separation by column chromatography. The only free amino groups in neocarzi-nostatin were succinylated completely in both derivatives. Amino terminal analysis, electrophoretic mobility and amino acid analysis were the same for both derivatives. However, only SUC-II was biologically active when assayed on three human cell lines and Sarcina lutea. Analysis by UV spectroscopy and circular dichroism indicated a difference in the molecular conformation, perhaps a slightly losened structure in SUC-I as compared with SUC-II. These isologous derivatives with different biological and optical activity have also been observed in neocarzinostatin. SUC-I seems to be a counterpart of "pre-NCS", an inactive form of NCS. SUC-II differs from NCS in being completely devoid of cytolytic and cytocidal activity. The retention of biological activity in bis-N-succinyl neocarzinostatin, in which all amino groups are bolocked, indicates that in NCS free amino groups are not essential for activity.