Molecular biology is reaching new depths in our understanding of the development and physiology of Malpighian tubules. In Diptera, Malpighian tubules derive from ectodermal cells that evaginate from the primitive hindgut and subsequently undergo a sequence of orderly events that culminates in an active excretory organ by the time the larva takes its first meal. Thereafter, the tubules enlarge by cell growth. Just as modern experimental strategies have illuminated the development of tubules, genomic, transcriptomic, and proteomic studies have uncovered new tubule functions that serve immune defenses and the breakdown and renal clearance of toxic substances. Moreover, genes associated with specific diseases in humans are also found in flies, some of which, astonishingly, express similar pathophenotypes. However, classical experimental approaches continue to show their worth by distinguishing between -omic possibilities and physiological reality while providing further detail about the rapid regulation of the transport pathway through septate junctions and the reversible assembly of proton pumps.