Phosphorothioate oligonucleotides with certain sequences or structure motifs can stimulate the immune system. We administered to mice a 27-mer phosphorothioate oligonucleotide (sequence 5'-TCG TCG CTG TCT CCG CTT CTT CTT GCC-3'), which has previously been shown to cause splenomegaly and hypergamma-globulinemia on in vivo administration in mice, and studied the pattern and kinetics of cytokine production at both the splenic mRNA and serum protein levels. Following i.p. administration of 50 mg/kg of oligonucleotide, significant increases in the splenic mRNA levels of IL-6, IL-12p40, IL-1 beta, and IL-1Ra and serum levels of IL-6, IL-12, MIP-1 beta, and MCP-1 were observed. In contrast, no significant differences in splenic mRNA levels of IL-2, IL-4, IL-5, IL-9, IL-13, IL-15, IFN-gamma, or MIF or serum levels of IL-2, IL-4, IL-5, IL-10, IFN-gamma, or GM-CSF were detected. The induction of IL-12 secretion was dependent on the sequence and dose of the oligonucleotides. One oligonucleotide (sequence 5'-GAG AAC GCT CGA CCT TCG AT-3') induced a high level of IL-12 secretion even at 5 mg/kg, whereas another oligonucleotide (sequence 5'-CTC TGC CAC CCA TCT CTC TCC TTC T-3') did not induce significant IL-12 secretion even at 50 mg/kg. IL-12 secretion induced by various doses of oligonucleotide has the same kinetics but differs in magnitude. These studies show a distinct pattern and kinetics of cytokine production following oligonucleotide administration and further demonstrate that cytokine induction is not a general property of phosphorothioate oligonucleotides but is dependent on the sequence and dose of the oligonucleotides.