Short-chain carboxylic acids (SCCA) are metabolic by-products of bacterial pathogens which can alter cytoplasmic pH and inhibit a variety of polymorphonuclear leukocyte (PMN) motile functions. Since cytoskeletal F-actin alterations are central to PMN mobility, in this study we examined the effects of SCCA on cytoskeletal F-actin. Initially, we tested nine SCCA (formate, acetate, propionate, butyrate, valerate, caproate, lactate, succinate, and isobutyrate). We document here that while eight altered cytoplasmic pH, only six altered cytoskeletal F-actin. We then selected one SCCA that altered both F-actin and cytoplasmic pH (propionate) and one SCCA that altered only cytoplasmic pH (lactate) for further study. Propionate, but not lactate, caused an irregular cell shape and F-actin distribution. Furthermore, propionate, but not lactate, inhibited formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated PMN polarization, F-actin localization, and cytoplasmic pH oscillation. Propionate-induced changes in cytoskeletal F-actin and cytoplasmic acidification were not affected by the fMLP receptor antagonist N-t-BOC-1-methionyl-1-leucyl-1-phenylalanine; however, alkalinization was affected. Pertussis toxin treatment completely inhibited propionate-induced changes in F-actin but had no effect on propionate-induced cytoplasmic pH oscillation. These results indicate that propionate (i) bypasses the fMLP receptor and G protein(s) to induce cytoplasmic pH oscillation, (ii) operates through G protein(s) to induce actin oscillation, cell shape changes (to irregular), and F-actin localization, and (iii) inhibits fMLP-stimulated cytoplasmic pH and actin oscillation, PMN polarization, and F-actin localization.