To clarify the pharmacological characteristics of Nalpha-amidino-Tyr-D-Arg-Phe-betaAla-OH (ADAB) and Nalpha-amidino-Tyr-D-Arg-Phe-MebetaAla-OH (ADAMB), mu1-opioid receptor-selective [D-Arg2]dermorphin tetrapeptide analogs, the plasma pharmacokinetics, and the in vivo blood-brain barrier (BBB) transport of these peptides were quantitatively evaluated. The mechanism responsible for the BBB transport of these peptides was also examined. The in vivo BBB permeation influx rates of 125I-ADAB and 125I-ADAMB after an i.v. bolus injection into mice were determined to be 0.0515 +/- 0.0284 microl/(min.g of brain) and 0.0290 +/- 0.0059 microl/(min.g of brain), respectively, both rates being slower than that of 125I-Tyr-D-Arg-Phe-betaAla-OH (125I-TAPA), a [D-Arg2]dermorphin tetrapeptide analog. To elucidate the BBB transport mechanism of ADAB and ADAMB, a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4) was used as an in vitro model of the BBB. The internalization of both 125I-ADAB and 125I-ADAMB into cells was concentration-dependent with half-saturation constant (Kd) values of 3.76 +/- 0.83 and 5.68 +/- 1.75 microM, respectively. The acid-resistant binding of both ADAB and ADAMB was significantly inhibited by dansylcadaverine (an endocytosis inhibitor) and poly-l-lysine and protamine (polycations), but it was not inhibited by 2,4-dinitrophenol, or at 4 degrees C. These results suggest that ADAB and ADAMB are transported through the BBB with slower permeation rates than that of TAPA, and this is likely to be a factor in the slow onset of their antinociceptive activity in the central nervous system. The mechanism of the BBB transport of these drugs is considered to be adsorptive-mediated endocytosis.