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Background: In parent-offspring communication, vocal signals are often used to attract attention and offspring might call to induce parental behaviour. In guinea pigs (Cavia aperea f. porcellus) mothers wean larger litters later than small ones, but it is unknown whether this difference depends on processes induced during pregnancy or is influenced post-natally by the number of pups present. We here tested with playback-experiments using pup separation calls whether mothers with cross-fostered large experimental litters (four-pup-litters) were more responsive to offspring calls and maintained responsiveness for longer than mothers with small experimental litters (two-pup-litters). Mothers were tested when two pups were suckling i.e. when both teats were occupied. Results: Mothers of four-pup litters responded stronger to broadcast pup separation calls than those with two-pup litters. Additionally, we tested the mothers' responsiveness to pup separation calls in the absence of their pups on day 8 and 20 of lactation. Mothers of four-pup litters responded stronger and showed no decrease in responsiveness from day 8 to 20, whereas mothers of two-pup litters responded less and decreased responsiveness from day 8 to 20. Mothers of four-pup litters also weaned their pups 5 days later than those of two-pup litters. Conclusion: Measured by their response to pup calls and by time to weaning, guinea pig mothers adjust maternal responsiveness to litter size. This behaviour is likely to be an adaptive strategy in resource allocation during reproduction.