We previously described the isolation of a variant subline of HL-60 cells that does not differentiate in response to nitric oxide (NO)-generating agents or to cGMP analogs. The variant cells have normal guanylate cyclase activity and normal NO-induced increases in the intracellular cGMP concentration. We now show that the variant cells have normal cGMP-dependent protein kinase (G-kinase) activity, both by an in vitro and in vivo assay, and using two-dimensional gel electrophoresis we have identified six G-kinase substrates in the parental cells. Of these six proteins, we found considerably less phosphorylation of one of the proteins in the variant cells than in parental cells, both in vitro and in intact cells, and by 35S-methionine/35S-cysteine incorporation we found much less of this protein in the variant cells than in parental cells. The protein is a shared substrate of cAMP-dependent protein kinase (A-kinase); since cAMP analogs still induce differentiation of the variant cells, it appears that the NO/cGMP/G-kinase and cAMP/A-kinase signal transduction pathways share some but not all of the same target proteins in inducing differentiation of HL-60 cells.