EBay has accused Amazon of using an "unlawful" scheme to tempt sellers on its auction site to move to the online retailer.
In a cease and desist letter, the auction site alleged that Amazon had used its internal messaging system to repeatedly contact traders.
EBay warned that it would take "appropriate steps" to protect itself from the tactic.
Amazon said it had started a "thorough investigation" of eBay's claims.
Details of Amazon's alleged activities emerged in a report published in the Wall Street Journal on Wednesday.
In that story, eBay said it found out in late September that Amazon staff were contacting sellers on the auction site via its messaging system.
Its investigation revealed that about 50 Amazon employees had sent more than 1,000 messages to regular traders, suggesting that they abandon eBay and instead sell via the retailer.
In its letter, eBay called the actions a "troubling scheme" and demanded that Amazon stop trying to poach merchants, reported Reuters.
Amazon's alleged actions contravene eBay's user agreements and might fall foul of Californian laws governing reasonable access to computer systems.