The mechanisms of apical (AP) uptake of cephalexin (CEPH) and loracarbef (LOR) in the absence or presence of an (extemally imposed) proton gradient were determined using well-stirred diffusion chambers that minimize the effects of the unstirred water layer. The results indicated that, compared to AP uptake in the presence of an imposed proton gradient, AP uptake in the absence of an imposed proton gradient had higher K(m) values and lower Jmax values. Furthermore, when inhibition studies were performed in the absence of a proton gradient, only natural peptides were effective, whereas the peptide analogs (e.g., enalapril) were not. In addition to the effects of concentration and competitive inhibitors, the results also indicated that (1) the AP uptake of both drugs was decreased more than 60% by FCCP, regardless of whether the proton gradient was present or absent; (2) effects of protein kinase C promoter were dependent upon the presence of a proton gradient; and (3) AP uptake in the presence of an imposed proton gradient was not affected by feeding restriction, whereas AP uptake in the absence of an imposed proton gradient was. These results showed for the first time that two substrates with similar AP uptake characteristics in the presence of an imposed proton gradient may not share those characteristics in the absence of an imposed proton gradient. Taken together, these results suggest that the AP uptake component that functions in the absence of an imposed proton gradient is distinctly different from the one that functions in the presence of an imposed proton gradient. Data generated from the present study and those in the literature lend support to the hypothesis that this distinctive component represents the second binding site on the AP peptide transporter. However, an alternative hypothesis that there are two AP peptide transporters remains to be disapproved.