The evolutionary dynamic of courtship signaling systems is driven by the interaction between male trait distributions and female preferences. This interaction is complex because females may choose mates based on multiple components of male signals, and female preference functions may vary depending on mate availability, female reproductive state, and environmental conditions. In Photinus fireflies (Coleoptera: Lampyridae), flying males emit bioluminescent flash signals to locate sedentary females, which reply selectively to attractive male flash signals with their own response flash. In this study, we first examined temporal variation in the paired-pulse flash patterns produced by Photinus greeni males in the field and found significant among-male variation (∼70% of total variation) in interpulse intervals (IPIs). There was no significant relationship between male IPI and spermatophore size, suggesting that P. greeni male courtship signals do not provide females with reliable indicators of male material resources. In laboratory playback experiments, we presented P. greeni females with simulated flash signals to assess how IPI and pulse duration independently affected the likelihood of female flash response. We also examined the effects of female body mass and time during the mating season on female preference functions, hypothesizing that females would be less discriminating when they were heavier (more fecund) and when mate availability declined. We found that P. greeni females discriminated among signals within their species' range based primarily on flash pattern IPI. Neither the time during the mating season nor female weight altered female preference functions for IPI, although season did influence female response to pulse duration. These results reveal that P. greeni females discriminate among conspecific males based primarily on male IPIs, the same signal character previously shown to be important for firefly species recognition. Field playback experiments indicated that female responsiveness peaked near the average IPI given by males at different ambient temperatures, suggesting that fireflies exhibit temperature coupling similar to that seen in many acoustically signaling animals.