(1) The marked inversion technique was used, and 38 major autosome sets of Drosophila melanogaster were extracted from populations of Jugoslavia, Taiwan and Japan. Spontaneous mutations were allowed to accumulate on the major chromosomes for 25 generations. Then the second chromosomes were tested to determine whether or not they were associated with such known mutator factors as the male crossing-over factor, SD, and the extrachromosomal element delta. Chromosome mutations were examined by salivary gland chromosome analysis, and viability mutations were done by the marked autosomal translocation method. (2) In 8 out of 38 major autosome lines, 5 inversions occurred in the second chromosomes, 4 inversions in the third chromosomes, and 1 reciprocal translocation between the second and the third chromosomes. Chromosome mutation rates were, therefore, 0.0063 per second chromosome per generation and 0.0053 per third chromosome per generation. Since there was no signficant difference in the rates, chromosome mutations seem to be occurring approximately equally in both major autosomes. (3) Lethal mutation rates were estimated to be 0.0097 per major autosome per generation. (4) Twenty-four second chromosome lines out of 37 demonstrated male crossing-over among the cinnabar and brown interval; the average frequencies were 0.0031 for all lines and 0.0034 when non-recombination lines were excluded. (5) One second chromosome exhibited delta retaining ability (ID), but no second chromosome carried SD.