Several transformation strains of Coprinopsis cinerea carry the defective tryptophan synthase allele trp1-1,1-6 which can be complemented by introduction of the trp1(+) wild-type gene. Regularly in C. cinerea, single-trp1(+)-vector transformations yield about half the numbers of clones than cotransformations with a non-trp1(+)-plasmid done in parallel. The effect is also observed with the orthologous Schizophyllum commune trpB(+) gene shown here to function as a selection marker in C. cinerea. Parts of single-trp1(+)- or single-trpB(+)-vector transformants are apparently lost. This paradoxical phenomenon relates to de-regulation of aromatic amino acid biosynthesis pathways. Adding tryptophan precursors to protoplast regeneration agar or feeding with other aromatic amino acids increases loss of single-trp1(+)-vector transformants and also sets off loss of clones in cotransformation with a non-trp1(+)-plasmid. Feedback control by tryptophan and cross-pathway control by tyrosine and phenylalanine are both active in the process. We deduce from the observations that more cotransformants than single-vector transformants are obtained by in average less disturbance of the tryptophan biosynthesis pathway. DNA in C. cinerea transformation usually integrates into the genome at multiple ectopic places. Integration events for a single vector per nucleus should statistically be 2-fold higher in single-vector transformations than in cotransformations in which the two different molecules compete for the same potential integration sites. Integration of more trp1(+) copies into the genome might more likely lead to sudden tryptophan overproduction with subsequent rigid shut-down of the pathway. Blocking ectopic DNA integration in a Delta ku70 mutant abolished the effect of doubling clone numbers in cotransformations due to preferred single trp1(+) integration by homologous recombination at its native genomic site.