Humans and other eukaryotic organisms manifest circadian (daily) rhythms which are controlled by an endogenous biochemical oscillator. Many cellular processes, including cell division, enzyme activity, and gene expression are timed by this oscillator. These "biological clocks" are important to human physiology. For example, psychiatric and medical studies have shown that circadian rhythmicity is involved in some forms of depressive illness, "jet lag," drug tolerance/efficacy, memory, and insomnia. Therefore, understanding the biochemical mechanisms of circadian clocks may lead to procedures which will be useful in the diagnosis and treatment of disorders which are relevant to sleep, mental health, and pharmacology. Despite the importance of clocked phenomena, however, the nature of the underlying biochemical mechanism remains elusive. Does it use or depend upon a known metabolic pathway, perhaps in a heretofore unsuspected way? Or is the circadian pacemaker driven by a totally unknown system? The biochemical mechanism of circadian oscillators is the most fundamental unanswered question in this field. The long-term objective of this research project is to clarify the cellular/molecular nature of circadian clocks in unicells. The strategic approach might be termed an analysis of the oscillator's inputs and outputs. The input pathway by which the clock is synchronized to the daily light/dark cycle will be identified by action spectroscopy, mutations, and pharmacology. The output pathways of the clock will be determined by a variety of techniques. For example, rhythmic proteins and mRNAs will be assayed by electrophoresis and nucleic acid hybridization. "Second messenger" output will be assayed by microelectrode recording, calcium-sensitive photoproteins, and radioimmunoassay of cyclic nucleotides. In addition, the possibility that these second messengers may be an integral part of the oscillator's mechanism will be tested by pharmacological analyses. Simple unicellular organisms offer technical advantages which should accelerate the identification of cellular processes involved in the circadian oscillators of all eukaryotes.