The macronucleus of the hypotrichous ciliate Oxytricha nova contains two DNA molecules with sizes of approx. 1.4 (actin II) and approx. 1.6 (actin I) kb that encode actin (Act) proteins. The 1.6-kb molecule has been previously described [Greslin et al., DNA 7 (1988) 529-536]. The 1.4-kb molecule is described here. The cloned molecule is 1393 bp in length, including 20 bp of telomere sequence at each end. It has an open reading frame (ORF) of 1119 bp that encodes a putative polypeptide of 374 amino acids (aa) designated as ActII. No introns are present. The coding segment is preceded at its 5' end by an A + T-rich 5' noncoding segment of 100 bp, excluding the telomere, and is followed by an A + T-rich 3' noncoding segment of 124 bp, also excluding the telomere. The aa sequence of the actII gene is 63% identical to actI of O. nova and 68, 70 and 69% identical to the rabbit, yeast and Tetrahymena Act aa sequences, respectively. ActI in O. nova is 64 to 68% identical to Act in other eukaryotes. Thus, the two Act in O. nova differ substantially from one another and neither conforms to the conserved Act sequence generally present in eukaryotes.