We have initiated a cytogenetic analysis of chromosome region 89A of Drosophila melanogaster by isolating a set of radiation-induced mutations causing loss of function of P[(w)B]1-1, a transposon bearing the white locus inserted in 89A. Complementation tests and cytological examination of these chromosomes identified four new deficiencies (Df(3R)Po2, Df(3R)Po3, Df(3R)Po4 and Df(3R)c(3)G2). The new deficiencies and three previously identified deficiencies (Df(3R)sbd26, Df(3R)sbd45 and Df(3R)sbd105) were tested for the ability to complement mutations in the enzyme loci Po and Aldox-1, the indirect flight muscle genes Tm2 and act88F, the morphological mutations jvl, sbd2 and Sb, the vital loci srp, pnr and mor, and a newly described vital locus l(3)89Aa. We also used linkage analysis to determine the order and relative positions of P[(w)B]1-1 and an independent transposon insertion, P[w+]21, with respect to cv-c, Po, Aldox-1 and sbd2. Cytological examination of the deficiencies and analysis of the transformed lines by in situ hybridization permits the correlation of genetically defined regions with specific polytene chromosome bands. A revised cytogenetic map of the 88F-89B region is presented.