The locus, Aph,for a third-instar larval alkaline phosphatase, 1-Aph, in Drosophila melanogasterhas been placed at 47.3 on the third chromosome. A new and fourth allele, Apha,is described. Another alkaline phosphatase, p-Aph, is characteristic of the pupal stage. Developmental studies show that 1-Aph begins to disappear 9.5 hr after prepuparium formation at 25 C, and that p-Aph appears as 1-Aph disappears. Each of the four Aph alleles produces a p-Aph variant distinguishable by electrophoresis. Except for the one produced by Apha,the electrophoretic properties of these p-Aph variants parallel those of the 1-Aph's produced by the same allele. Three sources of evidence support the conclusion that p-Aph variation is attributable to the Aphlocus: (1) In experiments where Aphalleles segregate, corresponding segregation of p-Aph variants is observed. (2) The linkage relationships of p-Aph are the same as those of Aph.(3) In experiments capable of detecting with a probability of 0.99 a recombination event between two loci 0.0006 centimorgans apart, no recombination is observed between 1-Aph and p-Aph. It is suggested that the Aphlocus either consists of one cistron which is responsible for both 1-Aph and p-Aph, or that it consists of two cistrons, one for 1-Aph and one for p-Aph. Implications for the structure of these alkaline phosphatases and for the nature of the developmental shift which they exhibit are discussed.