We have investigated the structural requirements for DNA cleavage by the isoschizomers HaeIII, BspRI and BsuRI which recognize the sequence ‐d(GGCC)‐. For this purpose decadeoxynucleotides were synthesized by the solid‐phase phosphotriester method and purified by high‐performance liquid chromatography. The kinetics of cleavage of these oligodeoxynucleotides were determined for the three isoschizomers with the following results.1. The sequence adjacent to the recognition site strongly influences the rate of cleavage. The preference is qualitatively the same for all three enzymes: AGGCCT > TGGCCA > GGGCCC ∼ CGGCCG, and follows the thermal stability of the different decanucleotides.2. Substitutions within the recognition site, namely dI for dG and dU for dC, affect the rate of cleavage differently for the three enzymes. The results can be rationalized in terms of an interaction of HaeIII with major and minor groove of the DNA, of BspRI mainly with the minor groove and of BsuRI with the major groove of DNA. It is obvious from our data that the mechanism of recognition of the same site is different for the three isoschizomers.