A synthetic 30-mer single-stranded oligodeoxyribonucleotide with a hexamer palindrome, AACGTT, induced IFN production and augmented NK activity in murine splenocytes. This effect does not appear to result from an antisense mechanism but rather is due to the palindrome. To clarify the required minimal size of the nucleotide, 10 kinds of 12- to 30-mer nucleotides were examined. Immunostimulatory activity of oligonucleotides 18 bases or more in length was observed and was proportional to the base length, with a maximum of 22-30 bases. On the other hand, the oligonucleotides 16 bases or less in length were not active even if they possessed the palindromic sequence. These results indicate that the immunostimulatory activity of oligonucleotides with certain palindromic sequences requires an oligonucleotide at least 18 bases long.