Numerous recombinants arose when protoplasts of S. coelicolor were treated with polyethylene glycol and regenerated on non-selective solid medium. In six-factor crosses, recombination frequencies of more than 10% (up to 17%) were routinely observed. This recombination did not require either of the known sex factors, SCPI and SCP2. The proportion of multiple crossover classes was much higher than amongst recombinants produced by conjugated between mycelia. Analysis of the spatial distribution of crossovers in double and quadruple crossover recombinants showed only a slight tendency for crossovers to occur closer together than randomly on the complete linkage group. This suggests that genomes brought together by protoplast fusion are complete, or nearly so (in conjugation, in contrast, one genome is represented by a comparatively short fragment). Individual colonies arising from fused protoplasts did not contain different parental genomes without recombinants, but recombinants often occurred without parentals. Several recombinant genotypes often occurred in the same colony, showing a segregation of some, only, of the parental alleles. Complementary genotypes, parental or recombinant, did not occur in the same colony. It is postulated that complete genomes of fused protoplasts usually become fragmented and that crossing-over, often repeated, occurs between the fragments, to generate haploid recombinants. Analysis of fusions between propoplasts of four different genotypes indicated that the average number of protoplasts fusing together was low, but nevertheless appreciable numbers of fusions involved three or four genomes. Crossing-over between them produced recombinants inheriting markers from three or four parents. The generation of nearly random populations of recombinants between two or more parent strains by propoplast fusion under the conditions described appears to have simple applications in industrial and academic strain construction.