Highly purified tryptic fragments of calmodulin were tested for their ability to stimulate adenylate cyclase activity of Bordetella pertussis spheroplast membranes and were compared to their activities on brain Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase. The C-terminal fragment, consisting of residues 78-148, was a full agonist for the cyclase with 0.1-0.15 the potency of calmodulin but did not stimulate phosphodiesterase. Fragments 1-77, 1-90, and 107-148 stimulated adenylate cyclase (and not phosphodiesterase) at low potency; this was not due to calmodulin contamination, but contamination by fragment 78-148 could not be excluded with certainty. An adduct of norchlorpromazine isothiocyanate and calmodulin showed full agonist activity for adenylate cyclase at 0.01-0.02 the potency of calmodulin. Stimulation of adenylate cyclase by a number of the fragments occurred in the absence of Ca2+, but stimulator potency was enhanced 20-60-fold in its presence. The similarity of Ca2+ requirements of fragment 78-148 and calmodulin suggests that occupancy of the two C-terminal Ca2+ binding sites of calmodulin accounts for most of the Ca2+ enhancement of calmodulin stimulation of adenylate cyclase.