From the Converse Chemical Laboratory, Harvard University, Cambridge, Massachusetts (Received for publication, April 24, 1959) In the preceding paper (1) we have investigated the metabolic basis for the sterol requirements of insects and have provided evidence to show that in the case of the beetle Dermestes vulpinus cholesterol biogenesis is multiply blocked. While the lack of sterol synthesis seems to be a metabolic defect common to all insects, the requirements for specific sterols vary widely among insect species. Cholesterol has been generally used as the dietary supplement and it supports growth and development equally well in all the insects examined. However most insects can be reared on sterols other than cholesterol as long as the sterols contain a fl-hydroxyl group at C-3 in the normal steroid ring system and a hydrocarbon side chain. Substituents at C-24 (as in plant and fungal sterols), double bonds in the side chain (stigmasterol and ergosterol), or even the lack of a nuclear double bond (cholestanol and tetrahydrostigmasterol) do not abolish biological activity, though they render the sterols less active in some cases. This generalization, however, holds only for insects which are omnivorous, herbivorous, or feed on plant products. For carnivorous insects such as the hide beetle D. vulpinus and the related Attugenus piceus only cholesterol and the closely similar ‘I-dehydrocholesterol and 24-dehydrocholesterol are satisfactory sterol sources. The results cited are the contribu- tions of many investigators and are summarized in several recent review articles (2-4). The objective of the present study was to ascertain whether the requirement for a cholesterol-type sterol by Dermestes is absolute. To this end various sterols which do not support the growth of Dermestes by themselves were tested for cholesterol- sparing activity. Underlying this approach was the hypothesis that the function of sterols in insects is 2-fold: (a) to provide the precursors for one or more metabolites which are themselves physiologically active (metabolic role), and (b) to serve as an essential cellular component without undergoing any metabolic alterations (structural role). Cholesterol, according to this hypothesis, can perform both of these functions. On the other hand, sterols capable of only partially replacing cholesterol for Dermestes might be able to perform one of these essential func- tions and thereby spare cholesterol. To test this possibility was the purpose of the experiments to be described. Support for the hypothesis of a dual function will be presented and it will * Supported by grants-in-aid from the United States Public Health Service, and Life Insurance Medical Research Fund, the National Science Foundation, and the Eugene Higgins Trust Fund of Harvard University. This work is taken from a thesis submitted by A. J. Clark to the Graduate School of Arts and Sciences of Harvard University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy. t Holder of a Predoctoral Fellowship from the National Science Foundation. become clear why, for these purposes, Dermestes is a singularly suited test organism. The metabolic role of cholesterol in was further examined by testing various steroids, known to be physiologically active in higher animals, for their ability to replace or spare cholesterol.