It is becoming increasingly clear that bistability (or, more generally, multistability) is an important recurring theme in cell signaling. Bistability may be of particular relevance to biological systems that switch between discrete states, generate oscillatory responses, or ''remember'' transitory stimuli. Standard mathematical methods allow the detection of bistability in some very simple feedback systems (systems with one or two proteins or genes that either activate each other or inhibit each other), but realistic depictions of signal transduction networks are invariably much more complex. Here, we show that for a class of feedback systems of arbitrary order the stability properties of the system can be deduced mathematically from how the system behaves when feedback is blocked. Provided that this open-loop, feedback-blocked system is monotone and possesses a sigmoidal characteristic, the system is guaranteed to be bistable for some range of feedback strengths. We present a simple graphical method for deducing the stability behavior and bifurcation diagrams for such systems and illustrate the method with two examples taken from recent experimental studies of bistable systems: a two-variable Cdc2͞Wee1 system and a more complicated five-variable mitogenactivated protein kinase cascade.O ne of the most important and formidable challenges facing biologists and mathematicians in the postgenomic era is to understand how the behaviors of living cells arise out of the properties of complex networks of signaling proteins. One interesting systems-level property that even relatively simple signaling networks have the potential to produce is bistability. A bistable system is one that toggles between two discrete, alternative stable steady states, in contrast to a monostable system, which slides along a continuum of steady states (1-9). Early biological examples of bistable systems included the lambda phage lysis-lysogeny switch and the hysteretic lac repressor system (10-12). More recent examples have included several mitogen-activated protein kinase (MAPK) cascades in animal cells (13-15), and cell cycle regulatory circuits in Xenopus and Saccharomyces cerevisiae (16)(17)(18). Bistable systems are thought to be involved in the generation of switch-like biochemical responses (13,14,19), the establishment of cell cycle oscillations and mutually exclusive cell cycle phases (17, 18), the production of self-sustaining biochemical ''memories'' of transient stimuli (20,21), and the rapid lateral propagation of receptor tyrosine kinase activation (22).Bistability arises in signaling systems that contain a positivefeedback loop (Fig. 1a) or a mutually inhibitory, doublenegative-feedback loop (which, in some regards, is equivalent to a positive-feedback loop) (Fig. 1b). Indeed, Thomas (23) conjectured that the existence of at least one positive-feedback loop is a necessary condition for the existence of multiple steady states; alternative proofs of this conjecture are given in refs. 24-27. However, the existence of positive loop...