An adaptive increase in the activity of the System A amino acid transporter is observed in various mammalian cell lines following prolonged amino acid (AA) deprivation. Recent publications from ourselves (Hyde et al. 2001) and other groups (Ling et al. 2001, Gazzola et al. 2001) have demonstrated that AA deprivation induces the increased expression of the sodium-coupled neutral amino acid transporter 2 (SNAT2), a functional System A transporter, at both the mRNA and protein level. The cellular events governing this response are poorly characterised, but a possibility is that amino acid-sensitive signal transduction pathways are modulated and lead to the altered expression of SNAT2. To address this possibility, we have studied System A adaptation in skeletal muscle cells in the presence of cell-permeable signal transduction inhibitors.Fully differentiated L6 myotubes were incubated in Earl′s balanced salt solution in the presence or absence of an AA mix at physiological concentration for 4 h. AA deprivation over this time course leads to an ∼ 4-fold increase in the transport of 10 µM [¹⁴C]-methylaminoisobutyrate, a selective System A substrate. A variety of structurally distinct kinase inhibitors were selected for use in signalling studies, these included PD-098059 (50 µM), rapamycin (100 nM), SB-203580 (10 µM), wortmannin (100 nM), genistein (100 µM) and SB-216763 (5 µM). Of these compounds, only wortmannin, genistein and SB-216763 were shown to significantly inhibit the adaptive regulation of System A (by 38 ± 11.2 %,59 ± 10.2 % and 50 ± 13.7 %, respectively; p < 0.05 in all cases, ANOVA). These results suggest that phosphoinositide-3-kinases, tyrosine kinases and glycogen synthase kinase-3 (GSK3), respective targets of the above three compounds, may play a role in amino acid signalling in L6 muscle cells.The potential involvement of GSK3 in System A adaptation is further indicated by similar effects of two additional GSK3 inhibitors, namely SB415286 and Li⁺ ions. Consistent with this, our recent experiments have demonstrated close parallels between the activities of glycogen synthase and System A in response to altered amino acid availability, indicative of a conserved amino acid sensing pathway with regulatory control over the two processes. The apparent involvement of GSK3 in this context is intriguing, since it has well documented roles in the control of glycogen and protein synthesis (Frame & Cohen, 2001).