The transport of thymidine by the protozoan parasite Giardia intestinalis was examined at 0 degrees C. This temperature prevented attachment of the cells to vessel walls, so that a rapid sampling technique could be used. Thymidine influx (distinguished from gross uptake) was readily measurable at 0 degrees C and was specific and saturable. The transporter appears to be a facilitative carrier, exhibiting a high affinity for thymidine (Km = 50 microM). Thymine and uracil were the most effective inhibitors (Ki = 30 microM and 45 microM, respectively), followed by thymidine, deoxyuridine and uridine (Ki = 64-96 microM). Cytosine, cytidine and deoxycytidine were not inhibitory, even at high concentrations. The data indicate that the oxygen at position 4 of the pyrimidine ring is essential for recognition by the transporter, whereas the 5-methyl group of thymine is unimportant. The furanose ring appears not to be recognized, since D-ribose was non-inhibitory and uridine and deoxyuridine were equally inhibitory but less so than uracil and thymine. This carrier probably mediates the transport of uracil, as well as uridine and thymidine, although influx of the base remains to be measured.