Artemia larval ribonuclease (Sebastián, J., and Heredia, C. F., (1978) Eur. J. Bichem. 90, 405-411) has been purified near homogeneity and its properties were studied. It consists of a single polypeptide chain of 38,000 daltons. It requires a divalent cation for activity. Ca2+ is the most effective among the metals tested. The metal dependence of the activity is biphasic. Maximal activity is obtained at 5-10 mM. In the absence of metals and chelating agents in the assay, 30-40% of the activity is observed. However, if chelating agents are added, the activity is abolished. At low concentrations of free metal (1-20 microM), 30-40% of maximal activity is obtained with Ca2+ or Mn2+, but not with Mg2+, Ca2+, but not Mn2+ or Mg2+, protects the enzyme from thermal inactivation. The best substrates for Artemia ribonuclease are poly(U) and poly(A), although with the latter it has only 10% the activity shown with the former. Using poly(U) as substrate, the products of a terminal digestion are P-2':3'-Urd and 3'-UMP. Using dinucleoside monophosphates as substrates, the enzyme is highly specific for a U residue at the 3' side of the phosphodiester bond (UpN), especially UpA, being inactive if the U residue is at the 5' side (NpU). Although some of its properties are similar to other eukaryotic or prokaryotic ribonucleases, its high specificity for UpN bonds suggest that this is a new type of ribonuclease. Moreover, it is a potentially useful enzyme for RNA analysis and/or sequencing.