Treatment of washed, ejaculated bovine sperm with 30 mM sodium fluoride immobilized the cells in a characteristically rigid form. In cells metabolizing endogenous substrates, fluoride decreased respiration by about 60%, but did not inhibit the cells’ ability to produce adenosine-5′-triphosphate (ATP) via oxidative phosphorylation and did not block access to endogenous substrates. Fluoride-immobilized sperm maintained maximal ATP titers for at least 60 min, but oligomycin treatment rapidly depleted ATP, indicating that ATP synthesis and metabolism was occurring in immobilized sperm. The putative phosphodiesterase inhibitor caffeine (2.5 mM) restored motility and increased respiration in fluoride-treated sperm, but 8-bromo-adenosine-3′,5′-monophosphate (8-bromo-cAMP) did not, even though 8-bromo-cAMP stimulated respiration in control (untreated) sperm. Carboxyfluorescein analysis of the intracellular pH of untreated sperm indicated a normal pH of 6.3. Fluoride addition decreased the apparent intracellular pH slightly, but this effect was attributable to dilution. Caffeine did not change internal pH in untreated or fluoride-immobilized sperm. Fluoride did not appear to affect cAMP metabolism, but caffeine increased intracellular cAMP titers by about 35% in both untreated and fluoride-inhibited sperm. However, caffeine treatment did not mimic 8-bromo-cAMP, as analyzed by electrophoresis and autoradiography of sperm proteins labeled with 32P from endogenously generated [32P]ATP. Clearly, caffeine is not stimulating motility in fluoride-treated sperm by affecting the cyclic AMP system. Fluoride also inhibited motility in digitonin-permeabilized sperm by a mechanism that may have involved magnesium depletion, but caffeine had no stimulatory effect on either untreated or fluoride-immobilized, permeabilized sperm.