Considerable experimental and theoretical effort has been exerted to understand how constant intersegmental phase relationships are produced between oscillators in segmentally organized pattern generating networks. The phase relationship between the segmental oscillators in the isolated timing network of the leech heartbeat central pattern generator is quite regular within individual preparations. However, it varies considerably among different preparations. Our goal is to determine how the phase relationships in this network are established. Here we assess whether inherent period differences, as suggested by the excitability-gradient hypothesis, play a role in establishing the phase relationships between the two coupled segmental oscillators of the leech heartbeat timing network. To do this we developed methods for reversibly uncoupling the segmental oscillators (sucrose knife) and pharmacological manipulation of the individual oscillators (split bath). Differences in inherent cycle periods between the third and fourth segmental oscillators (G3 and G4) were present in most (20 of 26) preparations. These period differences correlated with the phase differences observed between the segmental oscillators in the recoupled timing network, such that the oscillator with the faster cycle period, regardless of the segment in which it was located, led in phase in proportion to its period difference with the other oscillator. The phase differences between the original (coupled) and recoupled states of individual preparations were similar. Thus application and removal of the sucrose knife did not alter the period difference between the segmental oscillators in the timing network. Pharmacological manipulation of the uncoupled oscillators to alter the period difference between the oscillators led to similar correlated phase differences in the recoupled timing network. Across all experiments the uncoupled segmental oscillator with the faster cycle period established the cycle period of the timing network when recoupled. In conclusion, our findings indicate that an excitability-gradient plays a role in establishing the phase relationship between the segmental oscillators of the leech heartbeat central pattern generator since inherent period differences present between the oscillators are correlated to the phase relationships of the coupled/recoupled timing network.