H2ts125 is a fibre-defective, temperature-sensitive mutant of adenovirus serotype 2. H2ts125 fibre is unstable at the non-permissive temperature (ts phenotype), and does not migrate in the same way as the wild-type fibre in an SDS/polyacrylamide gel (elm phenotype). Sequence analysis has shown that H2ts125 carries two mutations on the fibre gene: Leu105 to Phe, and Ala434 to Val. Analysis of the structural modifications occurring in H2ts125 fibre was performed using peptide finger-printing and antipeptide sera as immunological probes. We found that all the detectable structural alterations in the mutant fibre were due to the substitution on codon 434. In addition, the ts phenotype was rescued by a wild-type DNA fragment containing the 3' moiety of the fibre gene and overlapping the 434th codon. Morphological analysis of fibre molecules observed under the electron microscope showed minor but statistically significant differences in the fibre length between mutant and wild-type. The mutant fibre was found to be slightly longer (308.8 +/- 1.9 A) than the wild-type fibre (300.1 +/- 2.1 A). Thus both ts and elm phenotypes were carried by the same Ala434 to Val mutation which probably resulted from a change in the three-dimensional structure of the fibre protein, and not from some proteolytic cleavage.