One hundred thirty-two isogenic lines for the O chromosome extracted from two natural Greek populations of D. subobscura, Parnes (P) and Crete (C), were used for this study, In these samples, we tested whether the observed heat-sensitive "alleles" were new hidden alleles of the tested loci A.O, M.E., and Xdh. The main conclusion from this experimental work is that the new, hidden, heat sensitive polymorphism does not belong solely to the tested locus, but is the result of interaction between the enzymatic products of different loci. The evidence for this is (1) that nonrepeatable heat-sensitive phenotypes within some tested strains exist, (2) The heat-sensitive phenotypes in F1 crosses did not give one pattern in most of the tested cases, and (3) in single testcrosses using null strains and single fly analysis, we did not observe one-locus Mendelian segregation within the offspring.