Plantaricin A (PlnA) is a 26-mer peptide produced by Lactobacillus plantarum C11. Although pheromone activity is a prime biological function of PlnA, the peptide also has membrane-permeabilizing strain-specific antibacterial activity. The possibility of membrane effects of PlnA on eukaryotic cells has previously not been studied in detail. We have therefore investigated the membrane-permeabilizing effects of PlnA on neoplastic (GH₄ cells) and normal (primary culture) rat anterior pituitary cells by means of microfluorometry (fura-2). The excitation light was switched between 360 and 380 nm. [Ca²⁺]_i was estimated from the ratio between emissions (510 nm) at the two excitation wavelengths (F360/F380), whereas the emission at the isosbestic wavelength 360 nm reflects the cytosolic fura-2 concentration. Within 5 s after exposure to 10-100 mM PlnA, [Ca²⁺]_i increased to saturating levels, followed by gradual loss of cytosolic fluorochrome during the subsequent 20-30 s. These results indicate a rapid and pronounced permeabilization of the membrane, even for organic molecules. The D-form of the peptide was as effective as the L-form, suggesting that PlnA exerts its membrane-permeabilizing effect through a non-chiral mechanism. Surprisingly, primary cell cultures of rat anterior pituitary were insensitive to 1 mM PlnA. These observations were supported by whole-cell and outside-out patch clamp recordings. 10-100 mM PlnA induced a rapid and unspecific conductance increase in GH₄ cells, whereas both normal pituitary cells and inside-out patches from GH₄ cells were insensitive to 1 mM PlnA. Thus, the peptide seems to differentiate both between plasma membranes and membrane leaflets.