We have previously reported that Catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC) activities, one soluble and one membrane associated. In this paper, the effect of neomycin and several divalent cations was analyzed, both in the soluble and the membrane-associated PLC activity in C. roseus transformed roots. In this system, neomycin, an aminoglycoside antibiotic, inhibited PLC in a concentration-dependent fashion. The neomycin IC50 (100 microM) was the same for the inhibition of the soluble and the membrane associated PLC activity. The effect of different divalent cations such as Ni2+, Cu2+, and Zn2+ was studied as well. In order to see the effect of these cations on PLC activity, we selected two conditions: a) in the presence of and b) in the absence of calcium. In the presence of calcium, these three divalent cations were able to inhibit PLC activity in both fractions in a concentration-dependent manner; however, the IC50s were different for the membrane and the soluble activities. For the soluble activity, the inhibition due to the three cations was very similar (IC50s between 0.2 and 0.3 mM). For the membrane associated PLC activity, Cu2+ was the most potent inhibitor (IC50 3.6 microM), then Ni2+ and then Zn2+. In the absence of calcium, higher concentrations of Cu2+ and Zn2+ demonstrated some inhibitory effect. We discuss the possible physiological role of these inhibitors on PLC activity.