The physical and biological properties of two new poly(U) analogs, poly(2-thiouridylic acid) or poly(s2U) and poly(2,4-dithiouridylic acid) or poly(s2s4U) have been investigated. Ultraviolet absorption and circular dichroism spectra of poly(s2U) and poly(s2s4U) as well as those of the corresponding monomers were measured. Absorption-temperature and absorption-pH profiles of both poly(s2U) and po1y(s2s4U) displayed sharp cooperative transitions. The phase transition diagrams of poly(s2U) and poly(s2s4U) show the high stability of their secondary structures.Neither poly(s2U) nor poly(s2s4U) formed helical complexes with poly(A). It was concluded from the corresponding phase transition diagrams that the reason for this behaviour is the larger thermodynamical stability of the poly(s2U) or poly(s2s4U) helices over the hypothetical complexes poly(s2U) poly(A) or poly(s2s4U) -poly(A). Sedimentation velocity analysis of poly(s2U) and poly(s2s4U) revealed sedimentation coefficients SZO, of 28 S and 12 S, respectively. A more detailed analysis of poly(s2U) lead to the conclusion that poly(s2U) exists as an intra-molecular associate of two anti-parallel poly(s2U) strands in the form of a large hairpin. Consistent with their physical properties, poly(s2U) and poly(s2s4U) are not able to replace poly(U) as messengers in the cell-free synthesis of poly(pheny1alanine). Poly(s2U) is only poorly, po1y(s2s4U) not a t all phosphoroIyzed by polynucleotide phosphorylase.I n the past years we have devoted our research interests to the chemistry and physico-chemistry of 4-thiopyrimidine nucleotides and their corresponding polynucleotides. Although 4-thiouridine was found to exist in syn conformation [l] in the crystalline state no evidence was obtained from nuclear magnetic resonance, optical rotatory dispersion or circular dichroism Bpectroscopy that s4UMP residues in polynucleotides adapt syn conformation [2,3]. The s4UMP residues in polynucleotides were found to be the Recently we reported the properties of a new poly(U) analog, poly(s2s4U). Poly(s2s4U) forms a very thermostable secondary structure and does not complex with poly(A) [lo]. Because poly(s4U) does not exhibit any secondary structure above 15°C we assumed that the high stability of the poly(s2s4U) structure might result from effects associated with 2-thioketo substitution in 2,4-dithiouracil residues. We therefore extended our studies to a series of polynucleotides containing s2CMP or s2UMP residues. The properties of a new poIy(C) analog poly(s2C) were described in recent contributions [ll, 121. A description of the synthesis and of the physical and biochemical behaviour of poly(s2U) and po1y(s2s4U) is the concern of this paper. The polymerisation of s2UDP and s2s4UDP to poly(s2U) or poly(s2s4U)