There are two forms of ornithine decarboxylase with respect to pyridoxal 5'-phosphate (pyridoxal-P) affinity in exponentially-growing Swiss 3T3 mouse fibroblasts: form I (Km approximately 10 muM) accounts for 30% of the total activity, and form II (Km approximately 0.4 muM) the remainder. Each form of the enzyme is in rapid equilibrium with ornithine and pyridoxal-P; neither form recognizes the Schiff base between ornithine and pyridoxal-P as a substrate. Total pyridoxal-P concentrations indicate that both forms may normally be at least partially active in vivo. Upon stimulation of 3T3 cells by pituitary growth factors, form I becomes undetectable within 4 h. As total activity increases over 10-fold during this time and continues to increase thereafter, a possible conversion of form I to form II could account for this increase only if the Km change reflects other changes in preexisting enzyme. The rates of cofactor dissociation are apparently the same for each form and neither rate changes with the growth state. Since rapid equilibrium kinetics apply, the forms apparently differ in their rate of cofactor association. The half-lives of the two forms in vivo are the same in unstimulated cells when measured concurrently. Also, the half-life of total activity decreases markedly upon stimulation as form II becomes dominant. These and other observations are not consistent with pyridoxal-P serving a major protective function for the enzyme in vivo.