Because most cancer deaths result from disseminated disease, understanding the regulation of tumor invasion and metastasis is a central theme in tumor cell biology. Interactions between extracellular matrices (ECM) and cellular microenvironment play a crucial role in this process. We have tested selected amino acids and polyamines for their ability to regulate RL95-2 cell invasion through both intact human amniotic basement menmbrane and a novel human ECM (Amgel). Three major systems for neutral amino acid transport, systems L, A, and ASC, are operational in these neoplastic cells. Amino acids entering the cell via transport system A or N, i.e., (methyl amino)-isobutyrate (MeAIB) or Asn, markedly enhanced invasiveness of these human adenocarcinoma cells as measured by a standard 72-hr amnion or Amgel invasion assay. Addition of 2-amino-2-norborane carboxylic acid (BCH; 1 mM), a model substrate of the L transport system, caused a significant decrease in invasive activity when tested in the Amgel assay. Interestingly, Val lowers steady-state levels of MeAIB uptake and blocks the increase in cell invasion elicited by MeAIB. At the same time, these amino acids do not influence cell proliferation activity. Neither the charged amino acid Lys or Asp (not transported by A/N/L systems) nor the polyamines putrescine, spermidine, or spermine modulate invasiveness under similar experimental conditions. Moreover, the observed time-dependent stimulation of system A activity (cellular influx of MeAIB) by substrate depletion is prevented by the addition of actinomycin D (5 μM) or cycloheximide (100 μM), suggesting the involvement of de novo RNA and protein synthesis events in these processes. MeAIB treatment of tumor cells selectively increased the activities of key invasion-associated type IV collagenases/gelatinases. These results indicate that in the absene of defined regulators (growth factors or hormones), certain amino acids may contribute to the epigenetic control of human tumor cell invasion and, by extension, metastasis. We propose that amino acids, acting via specific signaling pathways, modulate henotypic cell behavior by modulating the levels of key regulatory enzymatic proteins.