Secretion proteins from larval salivar glands of Drosophila melanogaster were analyzed with acrylamide gel electrophoresis. Four fractions were found; three showed electrophoretic variants in different wild-type stocks. Crossbreeding and cytogenetic techniques were used to localize the genes responsible for the two main fractions: The gene for fraction 3 was found to lie within a segment of the third chromosome which includes section 68C; the gene fo'r fraction 4, Sgs-4, was found to lie within section 3C8-3D1 of the X chromosome (1 -3-5). The puffs within these sections of the giant chromosomes are active before and during secretion synthesis and become inactive as secretion synthesis ceases. Larvae of one wild-type stock which lack protein fraction 4 do not exhibit any puffing in 3C. The relative amount of protein 4 in the salivary secretion shows a marked dependence on the dosage of the Sgs-4 gene in both duplication and deficiency genotypes. The active site within puff 3C11-12 apparently contains the structural gene for protein 4.A direct correlation between chromosomal sites which are transcriptionally active and activities in the cytoplasm has been established for puffs of the Balbiani ring type in the giant chromosomes of the salivary glands of Chironomus (1-3) and Acricotopus (4). In Drosophila melanogaster until now only indirect evidence suggesting the existence of such a relationship has been available (5-7). In the work on Drosophila melanogaster to be reported here we have now been able to obtain direct evidence of a cytogenetic nature indicating the location of the genes responsible for certain secretion proteins in individual salivary gland chromosome puffs. Furthermore, the effect of changes in dosage of one of the genes involved has been investigated. (5) y w spl cv f, X chromosome markers which were used for recombination analysis. The salivary gland chromosome locus of w is at 3C2; that of spl, at 3C7; and that of cv, between 4F1-2 -5D1-2. (6) Df(1)N8/dl-49, y Hw m2g4. In the X chromosome carrying Notch-S the section from 3C1 through 3D6-E1 is lacking. The chromosome is kept balanced with the inversion chromosome In(l )delta-49, marked with y HW m2g4. Df(1)scJ4R. All females carry attached X chromosomes and in addition they can carry the free duplication Dp(l;f)z9 of the X chromosome, i.e., section lAl through 3E7-F1;19-20. The males of this stock have an X chromosome with the deficiency Df(1 )SCJ4R, i.e., the section lB to 3A3-C2. The deficiency is compensated by the duplication Dp(lf)z9. (10) Cy/Pm;CxD/Sb. Both second chromosomes carry inversions; one is marked with Cy and one with Pm. One third chromosome carries an inversion and is marked with CxD; the other third chromosome has the normal structure and is marked with Sb.For more information on the genetic markers and their respective salivary chromosome sections see ref. 8. Linkage data for some X chromosome markers are given in Fig. 4.Preparation of the Salivary Glands and Their Chromosomes. The glands of late third instar larva...