Isatin-3-isonicotinylhydrazone was synthesized and characterized. The interaction of native calf thymus DNA with isatin-3-isonicotinylhydrazone (IINH) in 10 mM Tris-HCl aqueous solutions at neutral pH 7.4 has been investigated by spectrophotometric, circular dichroism (CD), melting temperature (T ( m )), spectrofluorimetric, and viscometric techniques. It is found that IINH molecules could intercalate between base pairs of DNA as are evidenced by: hypochromism in UV absorption band of IINH, induced CD spectral changes, sharp increase in specific viscosity of DNA, and increase in the fluorescence of methylene blue (MB)-DNA solutions in the presence of increasing amounts of IINH, which indicates that it is able to release the intercalated MB completely. The binding constants of IINH-DNA complex at four different temperatures (277, 288, 298, and 310 K) were calculated to be 4.7 × 10(4), 2.2 × 10(4), 1.75 × 10(4) and 1.1 × 10(4) M(-1), respectively. Furthermore, the enthalpy and entropy of the reaction between IINH and CT-DNA showed that the reaction is enthalpy-favored and entropy- disfavored (∆H = -30.187 kJ mol(-1); ∆S = -20.46 J mol(-1)K(-1)) which are other evidences to indicate the IINH is able to be intercalated in the DNA base pairs.