Cloned hybrid cells, selected for their ability to secrete an IgG 2a immunoglobulin specific for single-stranded (ss) nucleic acids, were obtained by fusion of spleen cells from an unimmunized autoimmune MRL/lpr male mouse with nonsecreting myeloma cells (MOPC-21, line Sp2/0-Agl4). Designated MRss-1, this monoclonal antibody was (i) propagated by intraperitoneal injection of hybrid cells into pristane- treated, Balb/c mice, (ii) purified from the bulk of other proteins in ascites extracts by chromatography with DEAESephacel adsorbent, and (iii) radiochemically labeled via reductive methylation using NaB3H4 and formaldehyde. The binding of 3H-labeled antibody to immobilized (ssDNAagarose, calf thymus) or soluble (fd DNA) ssDNA was rapid and dependent upon ssDNA and ionic strength, but not hydrogen ion concentration. Optimal binding occurred in both low and intermediate salt concentrations (0.01-0.25 M NaCl), yet was completely abolished above 0.30 M NaCl. The presence of guanine (Gua)-containing mono-, oligo-, and polynucleotides also abolished and/or decreased 3H-labeled antibody binding to ssDNA-agarose. In these competition assays, the amount of Gua-containing mono- and oligonucleotides required to inhibit antibody binding by 50% (0.2-1.0 mg/mL) exceeded those of poly(G), rRNA, and fd DNA (i.e., 0.03-0.1 µg/mL) by 4 orders of magnitude. In contrast, (deoxy)ribose 5'-phosphate as well as other nucleic acid derivatives devoid of Gua failed to inhibit antibody binding. The above findings were substantiated by the observation that 3H-labeled antibody bound to guanosine (G)- and guanidylate (pG)-conjugated Sepharose, yet not to other nucleoside (A, C, and U)- or nucleotide (pA, pC, and pU)-conjugated adsorbents. Last, the introduction of a methyl group at the N-2, O-6, and N-7 positions in the Gua ring system completely abolished antibody binding. Collectively, these results demonstrate that the MRss-1 antibody recognized single-stranded nucleic acid substrates by virtue of their content of guanidylate residues and, more specifically, by the presence of the Gua base moiety.