The present study sets out to investigate variation due to gender, F0, and/or prosodic position in Santa Ana del Valle Zapotec (Oto-Manguean), a language with phonemically breathy, modal and creaky vowels, each associated with a tone. Male and female speakers produced words in five prosodic positions: isolation (with focus, F0 higher than sentence-medial position), initial (focused, high F0), isolation (without focus, mid-range F0), medial (mid-range F0), final (lower F0). Two acoustic measures of phonation, H1-H2 and H1-A3, were made for each vowel. Results were inconclusive as to whether one gender was creakier or breathier than the other, though they did suggest that there was a difference in the production of phonation. In addition, there was also a strong effect of F0 on phonation, but not of position independently of F0. While the three-way phonation contrast was present in all five prosodic positions, it was not always well-defined. The contrast was minimized in isolation with focus (high F0) and initial position (high F0). The results obtained indicate that there is variation in phonation, even in a language with contrastive phonation.