In mice and humans, expression of the tumour necrosis factor receptor-1 (TNF-R1) gene in placental trophoblast cells is constitutive whereas expression of the TNF-R2 gene is developmentally programmed. In order to study the individual functions of TNF-R1 and -R2 in this lineage, cell lines were generated from placental explants of homozygous matings of gestation day 10 outbred mice (Swiss–Webster), TNF-R1-deficient (TNF-R1−/−) and TNF-R2−/−transgenic mice as well as the background strain for the TNF-R2−/−mice (WT, C57BL/6×129). All of the cells exhibited trophoblast markers; they contained cytokeratin intermediate filaments, expressed alkaline phosphatase activity and displayed transferrin receptors, but were negative for vimentin filaments and the macrophage marker, F4/80. Analysis of DNA by polymerase chain reaction demonstrated the expected TNF-R genotype in each line. In experiments testing the effects of recombinant mouse TNF-α (rmTNF-α) on viability and proliferation of the cell lines, rmTNF-α modestly but dose-dependently inhibited the growth of WT and TNF-R2−/−cells while having no effect on TNF-R1−/−cells. Actinomycin D-treated WT and, to a lesser extent, TNF-R2−/−cells, were more sensitive to growth inhibition than untreated cells whereas TNF-R1−/−cell responses remained unchanged. These data indicated that rmTNF-α inhibits growth of trophoblastic cells through TNF-R1 and that newly synthesized protein(s) provide partial protection against toxicity. In contrast to the receptor species-specific effects on cell growth exerted by rmTNF-α, both TNF-R mediated inhibition of alkaline phosphatase activity. Collectively, the observations support the postulate that receptor expression is the key factor which determines the nature and extent of TNF-α effects on trophoblast cell growth and function.