Poly(7-deazaguanylic acid) was enzymatically synthesized by the polymerization of 7-deazaguanosine 5''-diphosphate with polynucleotide phosphorylase from Micrococcus luteus in high yield. The homopolymer showed a similar thermal and total hypochromicity to poly(G) at the long wavelength absorption maximum. No sigmoid melting profile was observed for poly(c7G) [c7G = 2-amino-7-(.beta.-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidin-4-one] as is found for poly(G), implying a single-stranded structure in aqueous solution. From the circular dichroism spectra it can be concluded that the 7-deazapurine nucleotide is much more flexible than the purine nucleotide. In analogy to poly(G), the homopolymer poly(c7G) forms a 1:1 complex with poly(C) under neutral conditions, melting at a similar temperature to the poly(G) complex. At pH 2.5, where a poly(G).cntdot.2 poly(C) complex is observed, poly(c7G) still binds only 1 poly(C) strand. This is due to the lack of N-7 in poly(c7G), not allowing Hoogsteen base pair formation, which occurs with poly(G). RNase T1 cleaves poly(c7G), indicating that N-7 of guanosine is not a requirement for nucleotide binding to the enzyme, as has been suggested. Because of the single-stranded structure of poly(c7G), the polynucleotide chain is rapidly hydrolyzed by the single-strand-specific nuclease S1, where multistranded poly(G) is completely resistant.