This study examined the accumulation and transport of two related systemic opioids used as antidiarrhoeal drugs and compared their rates of transport with known P-glycoprotein (P-gp) substrates used in our in vitro environment. Cellular uptake and efflux and transcellular transport were all determined using Caco-2 cells after exposure to loperamide or diphenoxylate, with or without a range of efflux inhibitors. Bidirectional transport studies of 5 microM loperamide showed efflux to be fivefold higher than influx (42 x 10(-6) compared to 8 x 10(-6) cm/s); however, this decreased to twofold at 10 microM and was abolished using 100 microM loperamide. An uptake pathway was also discovered when P-gp was inhibited which, in the presence of Ca(2+) channel blockers, was amplified, providing a potential mechanism for central nervous system effects to be increased upon blockage of L-type calcium channels, quite separate from any P-gp inhibition. Diphenoxylate transport, however, showed little sign of P-gp-mediated efflux. Diphenoxylate accumulated readily within cells, yet transport through cells was very low. Additionally, efflux inhibitors had little impact on transport or accumulation, suggesting that diphenoxylate was not a substrate for an efflux mechanism.