Staff on South Western Railway (SWR) have begun a five-day strike over the future role of guards on trains.
Members of the National Union of Rail, Maritime and Transport Workers (RMT) stopped work at midnight and are due to return on Sunday.
SWR said it expected to run about two-thirds of normal services on weekdays and half of services on Saturday.
It is set to be the longest strike in the two-year dispute – the two previous lengthiest walkouts lasted three days.
SWR operates services from London Waterloo to Berkshire, Surrey, Hampshire, Dorset, Devon, Somerset and Wiltshire.
The rail operator said some routes would have no trains or replacement buses.
SWR said the union was “cynically targeting hard-working commuters, families trying to enjoy the half-term holidays and sports fans with its latest strike dates”.
In a statement, the firm said: “We have guaranteed a guard to be rostered on every single service, and our growth plans mean more guards, not fewer.”
RMT general secretary Mick Cash said the firm was looking to “open up a loophole that would allow them to run services driver-only at their discretion”.
He said: “There’s a simple solution to this dispute and it means SWR stop playing with words and negotiate the guard guarantee that… we have successfully negotiated in both Wales and Scotland and on a number of English franchises.”
Three-day strikes have previously been held on Southern Railway in August 2016 and on SWR from 31 August to 2 September.
The RMT has previously announced it will hold 24-hour strikes on SWR on each Saturday in November.