A visiting bank note collector was surprised to have the money rejected during two trips to Scotland.
RBS has been the only Scottish bank to issue the notes since 1989, but few are in circulation and they are rarely seen.
Its £1 notes are still legal currency but are normally only available from branches. RBS said they remained popular for gifts and weddings.
Surrey-based Gareth David said he made a point of using £1 notes when in Scotland.
However, booking office staff would not take them at Mallaig station last month.
That followed the notes being refused by Edinburgh Trams in February last year.
Mr David said: “I am a regular visitor to Scotland, and when I am in the capital I always visit the St Andrew Square branch of the Royal Bank of Scotland to withdraw some £1 notes for spending during my stay.
“As a banknote collector, I very much enjoy having the chance to spend £1 notes – something we have not be able to do in England for the past 30 years.
“The great majority of retailers are fascinated to see a £1 note, and for many it is the highlight of their day and they say they will save the notes to show their family and friends.
“I am on what increasingly feels like a one-man crusade to keep the £1 note in circulation.
“It was therefore very disappointing to encounter a number of people – including the ScotRail booking office at Mallaig – who flatly refused to accept the £1 notes, despite my polite insistence they remained in circulation and were still issued by RBS.
“Sadly, there seem precious few people left who will ask for the £1 notes in an RBS branch, so awareness of their remaining legal currency falls.”
Mr David complained to ScotRail, which told The Scotsman: “We are sorry to the customer the £1 note was not accepted. This was an error.
“We accept and bank any genuine Scottish notes and we have issued a reminder to all relevant staff.”
A spokesperson for Edinburgh Trams, who also apologised, said: “We’re a £1 note-friendly tramway, therefore we would accept notes issued by RBS. However, the notes are rarely seen in cash transaction, so our customers may find our staff a little more inquisitive than normal.”