Bacteriophages of the Podoviridae family use short noncontractile tails to inject their genetic material into Gram-negative bacteria. In phage P22, the tail contains a thin needle, encoded by the phage gene 26, which is essential both for stabilization and for ejection of the packaged viral genome. Bioinformatic analysis of the N-terminal domain of gp26 (residues 1-60) led us to identify a family of genes encoding putative homologues of the tail needle gp26. To validate this idea experimentally and to explore their diversity, we cloned the gp26-like gene from phages HK620, Sf6 and HS1, and characterized these gene products in solution. All gp26-like factors contain an elongated alpha-helical coiled-coil core consisting of repeating, adjacent trimerization heptads and form trimeric fibers with length ranging between about 240 to 300 A. gp26 tail needles display a high level of structural stability in solution, with T(m) (temperature of melting) between 85 and 95 degrees C. To determine how the structural stability of these phage fibers correlates with the length of the alpha-helical core, we investigated the effect of insertions and deletions in the helical core. In the P22 tail needle, we identified an 85-residue-long helical domain, termed MiCRU (minimal coiled-coil repeat unit), that can be inserted in-frame inside the gp26 helical core, preserving the straight morphology of the fiber. Likewise, we were able to remove three quarters of the helical core of the HS1 tail needle, minimally decreasing the stability of the fiber. We conclude that in the gp26 family of tail needles, structural stability increases nonlinearly with the length of the alpha-helical core. Thus, the overall stability of these bacteriophage fibers is not solely dependent on the number of trimerization repeats in the alpha-helical core.