Phytosulfokine-alpha [PSK-alpha, Tyr(SO(3)H)-Ile-Tyr(SO(3)H)-Thr-Gln], a sulfated mitogenic peptide found in plants, strongly promotes proliferation of plant cells in culture at very low concentrations. Oryza sativa PSK (OsPSK) cDNA encoding a PSK-alpha precursor has been isolated. The cDNA is 725 base pairs long, and the 89-aa product, preprophytosulfokine, has a 22-aa hydrophobic region that resembles a cleavable leader peptide at its NH(2) terminus. The PSK-alpha sequence occurs only once within the precursor, close to the COOH terminus. [Ser(4)]PSK-alpha was secreted by transgenic rice Oc cells harboring a mutated OsPSK cDNA, suggesting proteolytic processing from the larger precursor, a feature commonly found in animal systems. Whereas PSK-alpha in conditioned medium with sense transgenic Oc cells was 1.6 times as concentrated as in the control case, antisense transgenic Oc cells produced less than 60% of the control level. Preprophytosulfokine mRNA was detected at an elevated constitutive level in rice Oc culture cells on RNA blot analysis. Although PSK-alpha molecules have never been identified in any intact plant, reverse transcription-PCR analysis demonstrated that OsPSK is expressed in rice seedlings, indicating that PSK-alpha may be important for plant cell proliferation both in vitro and in vivo. DNA blot analysis demonstrated that OsPSK homologs may occur in dicot as well as monocot plants.