A partly purified calf spleen extract which inhibits lymphocyte proliferation in a tissue-specific, species-nonspecific, nontoxic, and reversible way, and may thus contain a chalone-like activity, has been further characterized. Our results show that the inhibitor works in G1-phase. The activity is resistant to heat, trypsin- and ribonuclease-treatment. It appears to be an acidic molecule and it does not appear to contain ribonucleic acid. These properties are compared to the characteristics which have been described for tissue-specific inhibitors of lymphocyte proliferation by other authors.