The chloroplast ribosomal intron of Chlamydomonas reinhardtii encodes a sequence-specific DNA endonuclease (I-CreI), which is most probably involved in the mobility of this intron. Here we show that I-CreI generates a 4 bp staggered cleavage just downstream of the intron insertion site. The I-CreI recognition sequence is 19-24 bp in size and is located asymmetrically around the intron insertion site. Screening of natural variants of the I-CreI recognition sequence indicates that the I-CreI endonuclease tolerates single and even multiple base changes within its recognition sequence.