We have used radiation inactivation analysis to determine the molecular size in situ of the system-L amino acid transporter in human erythrocytes. Erythrocytes can be frozen at 196°C in the presence of cryoprotective agents (280g of glycerol, 28g of sorbitol in 720ml of 150rnw-NaC1 and 20mM-dithiothreito1, 30min incubation, on ice) and reconstituted successfully for flux studies. Samples were irradiated w.rth a high-energy (16 MeV) electron beam (2-10 MRad, dose rate 2.0 MRad/ min, lOcm uniform beam) in evacualed glass ampoules (O.Sml, 50% haematocrit). Carrier-mediated L-[U 14-C]leucine uptake (extracellular concentration 0.2mw, 37°C) was measured by the method of Young &. Ellory (1982) using an incubation time of 30s to measure initial rates of transport (in the presence and absence of 50mw-non-radioactive L-phenylalanine to define the L-system). The freezing-thawing procedure had no deleterious effects on either K , or V,,,, for L-leucine influx via system-L(6.0m~ and 114mmol/litre of cells per h, respectively) in agreement with previous estimates in fresh cells (Young et al., 1980). The target size of the transport function was calculated directly from flux inactivation, as increasing amounts of radiation were found to inhibit carrier-mediated (Lphenylalanine-sensitive) L-leucine uptake in a dose-dependent manner, obeying single-hit target itheory (Fig. 1). The inactivation curve is the best fit line computed by a nonlinear regression program giving k = 0.0435 f 0.0056 for A = A,e+ (A, A, represent activity of dose D and zero dose), corresponding to a D 3 , of 23.OMRad. As an internal standard, G6PDH activity was assaye’d (Sigma Chemical Co.) and, also from Fig, 1, gave a k of 0.0520+0.0035 (D3 , 19.2MRad). The estimate of the target size of G6PDH using this k value and the empirical relationship M , = 6.4 x 10s/D3, (MRad) (Kepner (& Macey, 1968) is 33000 f 2200, a factor of 3.6 smaller than the reported M , of 120000 obtained by conventional methods (Levy, 1979). This reflects the effect of low temperature on radiation sensitivity in agreement with the work of Kempner & Haigler (1982) who reported an almost 4-fold reduction in target sizes calculated from data collected at 196°C compared with those obtained at + 30°C. A similar effect of temperature has been reported in a study of the target size of the nucleoside transporter, based directly on inactivation of uridine influx as well as ligand (nitrobenzylthioinosine) binding in cryoprotected human erythrocytes (Jarvis et al., 1984). Application of the temperature correction factor with the k of 0.0435 obtained for inactivation 01’ L-leucine transport gives a target size of 100000 f 13000 for system-L. in human erythrocytes. This is similar to that estimated for the