9-(2-Phosphonomethoxyethyl)adenine (PMEA) is an acyclic adenine nucleotide analog which exhibits potent and selective antiviral activity against herpesviruses and retroviruses. The study of [14C]PMEA uptake in HeLa S3 cells has shown that intracellular levels of the drug plateau after 1 h. Transport across the plasma membrane is saturable (concentration at half-maximal saturation [Kt], 0.39 microM; maximum rate of uptake [Vmax], 1.72 pmol/min.10(6) cells), and it can operate against the concentration gradient. Its significant dependence on temperature and on cellular density has been demonstrated. Following the treatment of cells with proteases, PMEA uptake strongly decreases. The transport process is considerably specific, since only a few phosphonate analogs act effectively as competitive inhibitors. Of these, 9-(2-phosphonomethoxyethyl)-2,6-diaminopurine (Ki = 0.24 microM) is the most efficient. Also, natural nucleotides competitively inhibit PMEA transport, depending on the nature of the nucleobase (thymine = adenine > guanine > cytosine < uracil) and on the position and number of phosphate groups. Nucleosides and nucleobases do not interfere with PMEA uptake. Cellular transport of adenosine and thymidine or uptake of AMP and ATP via conjugated activity of ectonucleotidases and nucleoside transporters is not affected by PMEA. By using vectorial labeling of plasma membrane proteins with Na125I combined with affinity chromatography, a 50-kDa protein which may mediate cellular transport of PMEA has been identified.